Hindawi Publishing Corporation

Advances in Bioinformatics
Volume 2014, Article ID 324753, 18 pages
http://dx.doi.org/10.1155/2014/324753

Research Article
Alternate Phosphorylation/O-GlcNAc Modification
on Human Insulin IRSs: A Road towards Impaired Insulin
Signaling in Alzheimer and Diabetes

Zainab Jahangir,1 Waqar Ahmad,2 and Khadija Shabbiri1,2

1
Department of Chemistry, GC University Lahore, Lahore, Pakistan
2
School of Biological Sciences, Goddard Building, The University of Queensland, Brisbane, QLD 4072, Australia

Correspondence should be addressed to Khadija Shabbiri; k shabbiri@yahoo.com

Received 24 August 2014; Accepted 10 November 2014; Published 17 December 2014

Academic Editor: Jian-Liang Li

Copyright © 2014 Zainab Jahangir et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Impaired insulin signaling has been thought of as important step in both Alzheimer’s disease (AD) and type 2 diabetes mellitus
(T2DM). Posttranslational modifications (PTMs) regulate functions and interaction of insulin with insulin receptors substrates
(IRSs) and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR) residues on IRSs.
Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine
(Ser) and threonine (Thr) residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and
promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation
on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated;
however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more
than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative
phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize
the risk of AD and T2DM.

1. Introduction by both inadequate insulin secretion and insulin resistance

Alzheimer’s disease (AD) is a neurodegenerative disease
associated with a progressive loss in memory and other
mental processes, resulting in dementia. In the AD brain,
some changes occur in tertiary structures of microtubule-
associated protein tau and 𝛽-amyloid (A𝛽) peptide result-
ing in self-association and deposition. Hyperphosphorylated
forms of tau act as major constituents of neurofibrillary
tangles (NFTs), while A𝛽 is major constituent of plaques [1].
By 2050, there is expected to be a million new cases of AD per
year, or one new case every 33 seconds and AD prevalence is
proposed to be 11 to 16 million [2]. On the other hand diabetes
mellitus (DM) is characterized by hyperglycemia. Type 1
diabetes is insulin-dependent diabetes mellitus caused by
absolute deficiency of insulin secretion while type 2 diabetes
(T2DM) is non-insulin-dependent diabetes mellitus caused
of target organs [3]. In DM, numerous peripheral organs,
such as kidneys, eyes, and peripheral nerves, undergo patho-
logical changes. DM also affects the central nervous system
(CNS). Specifically, both types of diabetes are associated
with impairment of learning abilities and memory deficits
[3]. Hoyer in 1998 presented a hypothesis stating that brain
insulin may be resisted in AD and that AD can be called
a brain specific “type 3 diabetes.” Both AD and T2DM are
degenerative diseases characterized by neuronal loss and
𝛽-cells destruction, respectively. Impaired insulin signaling
resulting in neurodegeneration and cognitive damages is
the main systematic link between these two diseases. Many
studies found that DM almost doubles the risk of AD. It
was also found that diabetes is not only the risk factor
of AD but it also accelerates the onset of AD. Recent
studies also elaborate the involvement of impaired insulin
2 Advances in Bioinformatics
signaling in tau hyperphosphorylation. AD and DM also
share several enzymes (DOPA decarboxylase, glutamic acid
decarboxylase) and growth factor receptors (thyrotrophin-
releasing hormone, neuronal growth factor receptors, and
p75 receptors) [4–9].
Insulin receptor substrate proteins (IRSs) act as interac-
tion platforms that are phosphorylated on multiple tyrosine
residues and attract different signaling proteins to spread
the stimulus. Two isoforms of IRSs, that is, IRS-1 and IRS-
2, are most abundant in mammals and have many tyrosine
phosphorylation sites. In general, IRS-1 is important for
growth, while proper function of pancreatic 𝛽-cells and
glucose homeostasis are main functions of IRS-2 [10, 11].
Knockout of either of them causes insulin resistance, whereas
only IRS-2-knockout results in diabetes. Either IRS-1 or IRS-2
gene deletion in mice leads to insulin resistance [12]. Irrecon-
cilably, heterozygous and brain specific deletion of IRS-1 and
IRS-2 in mice increases duration of life [13, 14]. However, IRS-
2 deletion is important to induce tau phosphorylation and it
also accumulates cytoplasmic deposits of phosphorylated tau
[15]. Loss of IRS-2 in neurons of AD patients is particularly
severe. IRS-1 and IRS-2 decreased levels in AD neurons
increase NFT pathology. Reduced IRS-1 and IRS-2 levels
indicate ineffective signaling of IR and IGF-1R in AD [16].
Apart from their important role in metabolic signaling, IRSs
also spread proliferative and antiapoptotic signals and as a
result become overexpressed in most cancers [17].
In biological systems, posttranslational modifications
(PTMs) determine protein activity, localization, and their
interaction with other proteins [18]. Multiple kinases acti-
vated by many triggers of insulin resistance, like reactive
oxygen species [19], inflammatory cytokines [20], or excess
lipids [19, 20], phosphorylate IRSs on several Ser/Thr residues
[20]. This phosphorylation on various Ser/Thr residues is
thought to inhibit insulin signaling and induce insulin resis-
tance by blocking IRSs Tyr phosphorylation. Increased levels
of inactivated phosphoserine-312-IRS-1 and phosphoserine-
616-IRS-1 associated with decreased levels of IRS-1 and IRS-
2 were identified in AD neurons. These increased levels of
inactivated phosphoserine are strongly colocalized with NFTs
[16]. Phosphorylation of Ser or Thr residue results in IRS-
1 or IRS-2 inactivation [21]. IRS-1 after Ser/Thr phosphory-
lation becomes a weak substrate for enzyme insulin recep-
tor tyrosine kinase [22–24]. Phosphoserine-312-IRS-1 causes
IRS-1 inactivation by disordering IRS-1 binding to insulin
receptor (IR). However, phosphoserine-616-IRS-1 prevents
insulin activation of PI3-kinase [21, 23, 25]. Mechanistically
many kinases that are overexpressed in AD phosphorylate
IRS-1 result in IRS-1 inactivation and IR/IGF-1R resistance.
Phosphorylation and O-glycosylation are also involved in
affinity changes of IGFBP-6 and cause inhibition of IGF-II
action on target cells [26, 27]. O-glycosylation of IGFBP-
6 leads to 10-fold higher affinity for IGF-II by preventing
IGFBP-6 binding to glycosaminoglycans and cell membrane
[28].
Analogous to phosphorylation, O-glycosylation is also
one of the important PTMs during which N-acetyl-glucos-
amine (O-GlcNAc) molecule is introduced on Thr or Ser
residue by O-GlcNAc transferase (OGT). Phosphorylation
on Thr or Ser residue can be inhibited by the addition of
O-𝛽-GlcNAc. Interplay between these two PTMs on the same
residues also known as yin yang sites has been reported in
several nuclear and cytoplasmic proteins [29]. These PTMs
regulate many functions of the proteins and are responsible
for temporary conformational changes [30]. Various studies
have shown that O-glycosylation on IRSs reduced in AD
and T2DM due to impaired insulin signaling and glucose
metabolism [31–34]. Although some Ser/Thr sites are mapped
to be O-glycosylation on C-terminal of IRSs, their exact
location is still undetermined. This work predicts potential
phosphorylation, O-𝛽-GlcNAc, and yin yang sites on IRS-
1 and IRS-2 proteins, involved in metabolic functions in
the brain by using various bioinformatics tools. Impaired
phosphorylation and O-glycosylation on IRS 1 and 2 lead
to defective insulin signaling that resulted in Alzheimer-
and diabetes-like complications. The sites where this type of
crosstalk occurs are useful targets for designing new drugs for
resulting diseases.
2. Materials and Methods
2.1. Multiple Alignment and Sequences Retrieval. The FASTA
sequences of human IRS-1 and IRS-2 were taken from the
Uniprot sequence database (http://www.uniprot.org/uniprot/
?query=irs&sort=score) with entry name IRS1 HUMAN and
accession number P35568 and entry name IRS2 HUMAN
with accession number Q9Y4H2, respectively. Blast of these
two sequences was made by Blast/Uniprot. A total of 37
hits for IRS-1 and 24 hits for IRS-2 were obtained with E-
value of zero or less. Sequences based on uncharacterized,
putative, hypothetical, predicted, and unnamed proteins were
neglected. For the multiple alignments of IRS-1 proteins,
eight sequences were selected from 37 retrieved sequences.
The accession numbers of these selected sequences are
given in Table 1. For the alignment of IRS-2 proteins, three
sequences were selected from 24 retrieved sequences. The
accession numbers of these three selected sequences are
given in Table 2. ClustalW (http://www.ebi.ac.uk/Tools/msa/
clustalw2/) was used for the multiple alignments of all the
sequences of IRS-1 and IRS-2 to get the conservation status
of serine, threonine, and tyrosine residues.
2.2. Protein Structure Prediction and Analysis. As there were
no complete 3D structures of human IRS 1 and IRS 2 available
in protein data bank, ab initio models were designed by using
software MODELLER (ModWeb: https://modbase.compbio
.ucsf.edu/scgi/modweb.cgi) [35], I-TASSER (http://zhanglab
.ccmb.med.umich.edu/I-TASSER/) [36], and Swiss Model
(http://swissmodel.expasy.org/) [37].
On the basis of Ramachandran plots, best ab initio models
were selected. Ramachandran plots were taken from Mol-
Probity (http://molprobity.biochem.duke.edu/) server [38].
FATCAT web server (http://fatcat.burnham.org/fatcat-cgi/
cgi/fatcat.pl?-func=pairwise) was used to perform pairwise
alignment of protein 3D structure. Chimera (http://www.cgl
.ucsf.edu/chimera/) [39] and Jmol (http://jmol.sourceforge
.net/) software were used to view and analyze 3D structure.
Advances in Bioinformatics 3

Table 1: Different IRS-1 proteins used for multiple alignment.

Species name Accession number Identity Score 𝐸-value

Human (Homo sapiens) P35568 100% 6,593 0.0
Green monkey (Chlorocebus aethiops) Q28224 96% 6,392 0.0
Pig (Sus scrofa) Q68H99 92% 6,016 0.0
Chicken (Gallus gallus) P79773 90% 5,854 0.0
Chinese hamster (Cricetulus griseus) G3IDC4 89% 5,804 0.0
Mouse (Mus musculus) Q543V3 89% 5,786 0.0
Rat (Rattus norvegicus) P35570 89% 5,783 0.0
Western clawed frog (Xenopus tropicalis) F6XDD6 59% 3,161 0.0

Table 2: Different IRS-2 proteins used for multiple alignment.

Species name Accession number Identity Score 𝐸-value

Human (Homo sapiens) Q9Y4H2 100% 7,108 0.0
Rat (Rattus norvegicus) F1MAL5 85% 5,908 0.0
Mouse (Mus musculus) P81122 85% 5,891 0.0

2.3. Prediction Methods for PTMs. We used three distinct
algorithms: support vector machine (SVM), neural network
(ANNs), and hierarchal searches [40] to reduce the false
negative and false positive sites during predicting PTM sites
on human IRS-1 and IRS-2. The NetPhos server generates
neural network predictions for Ser/Thr/Tyr phosphorylation
sites in eukaryotic proteins. The NetPhosK 1.0 produces
kinase specific phosphorylation sites predictions. KinasePhos
is a new version of kinase-specific phosphorylation site pre-
diction server. Phospho.ELM is a database of experimentally
confirmed phosphorylation sites in eukaryotic proteins. The
YinOYang server predicts O-𝛽-GlcNAc attachment sites in
eukaryotic nuclear and intracellular protein sequences. These
bioinformatics tools have been used efficiently in many
studies [27, 41, 42].
2.3.1. Phosphorylation Residues and Related Kinases Predic-
tion. Neural network-based programs NetPhos 2.0 (http://
www.cbs.dtu.dk/services/NetPhos/) [43] and Scansite (http://
scansite.mit.edu/motifscan id.phtml) [44] were used to pre-
dict phosphorylation potential for human IRS 1 and IRS 2
for serine, threonine, and tyrosine residues. 0.5 is minimum
threshold value for NetPhos 2.0 used to predict phosphoryla-
tion.
Prediction of kinase specific phosphorylation sites in
human IRS 1 and IRS 2 was made by Scansite (http://scansite
.mit.edu/motifscan id.phtml) [44], NetPhosK 1.0 (http://cbs
.dtu.dk/services/NetPhosK/) [45], and KinasePhos 2.0 (http://
kinasephos2.mbc.nctu.edu.tw/) [46]. Kinase specific accep-
tor substrates including Thr, Ser, and Tyr are predicted by
these programs.
Phospho.ELM (http://phospho.elm.eu.org) [47] and Uni-
prot (http://www.uniprot.org/uniprot/) databases were used
to obtain the data regarding experimentally verified phospho-
rylation sites on human IRS 1 and IRS 2.
2.3.2. O-Glycosylated Residues and Yin Yang Sites Prediction.
YinOYang 1.2 (http://www.cbs.dtu.dk/services/YinOYang/)
database [48] was used to predict O-𝛽-GlcNAc modification
potential sites. The potential phosphorylation sites can also
be predicted by YinOYang 1.2 program. Hence, this program
predicts the yin yang sites with highly uneven threshold
that is adjusted in accordance with amino acid surface
accessibility. This method also helps to predict the potential
yin yang sites. Modification potential and conservation status
of Thr and Ser residues were used to determine the false
negative (FN) yin yang sites.
The surface accessibility of predicted Ser, Thr, and Tyr
residues for posttranslational modifications was assessed by
using NetSurfP (http://www.cbs.dtu.dk/services/NetSurfP/)
[49]. Scansite (http://scansite.mit.edu/motifscan id.phtml)
[50] was also used to check surface accessibility of human IRS
1 and IRS 2 to solvents and for PTMs.
3. Results
3.1. Alignment of Sequences to Determine the Conservation
of Ser/Thr/Tyr Residues within IRS-1. To determine the evo-
lutionary conservation of Ser/Thr/Tyr residues, IRS-1 was
aligned with orthologous sequences of other species includ-
ing green monkey, pig, chicken, Chinese hamster, mouse, rat,
and western clawed frog (Figure 1). Out of 182 Ser residues,
99 (54.3%) were conserved, 12 (6.5%) were conserved sub-
stitute, and 29 (15.9%) were semiconserved. For total 60 Thr
residues, 29 (48.3%) were conserved, 9 (15%) were conserved
substitutes, and 5 (8.3%) were semiconserved. Same as for
total 32 Tyr residues where 25 (78.1%) were conserved and
1 (3.1%) was conserved substitution. This data showed that
human IRS-1 has high conservation status.
3.2. Alignment of Sequences to Determine the Conservation
of Ser/Thr/Tyr Residues within IRS-2. IRS-2 was aligned
with only known orthologous sequences of species rat and
mouse (Figure 2). Like IRS-1, human IRS-2 showed high
conservation among different species. Out of 177 Ser residues,
4 Advances in Bioinformatics

IRS1 HUMAN MASPPE---SDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYY 47

IRS1 CHLAE MASPPE---SDGFSDVRKVGYLRKPKSMHKRFFVLRAASETGDPARLEYY 47
IRS1 CHICK MASPPE---SDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGAPG-VEYY 46
IRS1 MOUSE MASPPD---TDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYY 47
IRS1 RAT MASPPD---TDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYY 47
IRS1 CRIGR MASPPD---TDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYY 47
IRS1 PIG -------------SDVRKVGYLRKPKSMHKRFFVLRAASEAGGPARLEYY 37
IRS1 XENTR MASPTDPQAQENFSDVRKVGYLRKPKSMHKRFFVLRSASES-GLARLEYY 49
∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ : ∗∗∗ : . : ∗∗∗

IRS1 HUMAN ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 97

IRS1 CHLAE ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 97
IRS1 CHICK ENEKKWWHKSSAPKRSIPLESCFNINKQVGDKNKHLVALYTRDEHFAIAA 96
IRS1 MOUSE ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 97
IRS1 RAT ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 97
IRS1 CRIGR ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 97
IRS1 PIG ENEKKWRHKSSAPKRSIPLESCFNINKRADSKNKHLVALYTRDEHFAIAA 87
IRS1 XENTR ENEKKWRHKSGAPKRSIPLESCFNINKRADSKNKHLVALYTKEECFAIAA 99
∗∗∗∗∗∗ ∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ : . . . ∗∗∗∗∗∗∗∗∗∗ : : ∗ ∗∗∗∗∗
IRS1 HUMAN DSEAEQDSWYQALLQLHNRAKGHHDGAAALGAGGGGGSCSGSSGLGEAGE 147
IRS1 CHLAE DSEAEQDSWYQALLQLHNRAKGHHDGAAALGAGGGGGSCSGSSGLGEAGE 147
IRS1 CHICK DSEAEQDSWYQALLPLHNRAKGHHDGAAALG-GGGGGSCSSSASLGEAGE 145
IRS1 MOUSE DSEAEQDSWYQALLQLHNRAKAHHD---GAGGG-CGGSCSGSSGVGEAGE 143
IRS1 RAT DSEAEQDSWYQALLQLHNRAKAHHD---GAGGG-CGGSCSGSSGVGEAGE 143
IRS1 CRIGR DSEAEQDSWYQALLQLHNRAKAHHD---GAGGGGCGGSCSGSSGVGEAGE 144
IRS1 PIG DSEAEQDSWYQALLQLHNRAKGHHDGAAGPGAGGGGGSCSGSSGLGEAGE 137
IRS1 XENTR ECEQEQDGWYQALVDLHNRGKTHHQ-------HHNHDGATNGVHDGLNGD 142
: . ∗ ∗∗∗ . ∗∗∗∗∗ : ∗∗∗∗ . ∗ ∗∗ : ... :. . ∗ ∗:
IRS1 HUMAN DLSYGDVPPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 197
IRS1 CHLAE DLSYGDVPPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 197
IRS1 CHICK DLSYGDGAPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 195
IRS1 MOUSE DLSY-DTGPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 192
IRS1 RAT DLSY-DTGPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 192
IRS1 CRIGR DLSY-DTGPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 193
IRS1 PIG DLSYGDVPPGPAFKEVWQVILKPKGLGQTKNLIGIYRLCLTSKTISFVKL 187
IRS1 XENTR DVYGEVTPPGLAFKEVWQVIMKPKGLGQLKNLVGIYRLCLTNRTISLVKL 192
∗: ∗∗ ∗∗∗∗∗∗∗∗∗ : ∗∗∗∗∗∗∗ ∗∗∗ : ∗∗∗∗∗∗∗∗ . : ∗∗∗ : ∗∗∗

IRS1 HUMAN NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 247

IRS1 CHLAE NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 247
IRS1 CHICK NSDAAAVVLQLLNIRRCGHSENFFFIEVGRSAVTVPGEFWMQVDDSVVAQ 245
IRS1 MOUSE NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 242
IRS1 RAT NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 242
IRS1 CRIGR NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 243
IRS1 PIG NSEAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGPGEFWMQVDDSVVAQ 237
IRS1 XENTR NSDAAAVVLQLMNIRRCGHSENFFFIEVGRSAVTGAGEFWMQVDDSVVAQ 242
∗∗ : ∗∗∗∗∗∗∗∗ : ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗
IRS1 HUMAN NMHETILEAMRAMSDEFRPRSKSQSSSNCSNPISVPLRRHHLNNPPPSQV 297
IRS1 CHLAE NMHETILEAMRAMSDEFRPRSKSQSSSNCSNPISVPLRRHHLNNPPPSQV 297
IRS1 CHICK NMHETILEAMRAMSEEFRPRSKSQSSSNCSNPISVPLRRHHINNPPPSQV 295
IRS1 MOUSE NMHETILEAMRAMSDEFRPRSKSQSSSSCSNPISVPLRRHHLNNPPPSQV 292
IRS1 RAT NMHETILEAMRAMSDEFRPRTKSQSSSSCSNPISVPLRRHHLNNPPPSQV 292
IRS1 CRIGR NMHETILEAMRAMSDEFRPRSKSQSSSSCSNPISVPLRRHHLNNPPPSQV 293
IRS1 PIG NMHETILEAMRAMSDEFRPRSKSQSSSNCSNPISVPLRRHHLNNPPPSQV 287
IRS1 XENTR NMHETILEAMKALSDEFRPRSKSQSSSNCSNPISVPLRRHHLNHPPPSQV 292
∗∗∗∗∗∗∗∗∗∗ : ∗ : ∗ : ∗∗∗∗∗ : ∗∗∗∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗ : ∗: ∗∗∗∗∗∗
IRS1 GLTRRSRTESITATSPASMVG--GKPGSFRVRASSDGEGTMSRPASVDGS
HUMAN 345
IRS1 GLTRRSRTESITATSPASMVG--GKPGSFRVRASSDGEGTMSRPASVDGS
CHLAE 345
IRS1 GLSRRSRTESVTATSPAGGVG--GKPSSFRVRESSDGEGTMSPPDQQDGS
CHICK 343
IRS1 GLTRRSRTESITATSPASMVG--GKPGSFRVRASSDGEGTMSRPASVDGS
MOUSE 340
IRS1 RAT GLTRRSRTESITATSPASMVG--GKPGSFRVRASSDGEGTMSRPASVDGS 340
IRS1 GLTRRSRTESITATSPASMVG--GKPGSFRVRASSDGEGTMSRPASVDGS
CRIGR 341
IRS1 PIG GLTRRSRTESITATSPASLVG--GKQGSFRVRASSDGEGTMSRPASVDGS 335
IRS1 GLNRRARTESVTATSPAGGVAKHGSSSSFRVRASSDGEGTMSRPASMEGS
XENTR 342
∗∗. ∗∗ : ∗∗∗∗ : ∗∗∗∗∗∗ . ∗ . ∗ . . ∗∗∗∗∗ ∗∗∗∗∗∗∗∗∗ ∗ . :∗∗
IRS1 HUMAN PVSPSTNRTHAHRHRGSARLHPPLNHSRSIPMPASRCSPSATSPVSLSSS 395
IRS1 CHLAE PVSPSTNRTHAHRHRGSARLHPPLNHSRSIPMPASRCSPSATSPVSLSSS 395
IRS1 CHICK PVSPSTNRTHAHRHRGSALLQPPLNHSRSIPMPASRCSPSATSPVSLSSS 393
IRS1 MOUSE PVSPSTNRTHAHRHRGSSRLHPPLNHSRSIPMPSSRCSPSATSPVSLSSS 390
IRS1 RAT PVSPSTNRTHAHRHRGSSRLHPPLNHSRSIPMPSSRCSPSATSPVSLSSS 390
IRS1 CRIGR PVSPSTNRTHAHRHRGSSRLHPPLNHSRSIPMPSSRCSPSATSPVSLSSS 391
IRS1 PIG PVSPSTNRTHAHRHRGSSRLHPPLNHSRSIPMPSSRCSPSATSPVSLSSS 385
IRS1 XENTR PVSPSASRAQSHRHRGSSRLHPPLNHSRSIPMPATRCSPSATSPVSLSSS 392
∗∗∗∗∗ : . ∗ : : : ∗∗∗∗∗∗ : ∗ :∗∗∗∗∗∗∗∗∗∗∗∗ : : ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

IRS1 HUMAN STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 445

IRS1 CHLAE STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 445
IRS1 CHICK STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 443
IRS1 MOUSE STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 440
IRS1 RAT STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 440
IRS1 CRIGR STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 441
IRS1 PIG STSGHGSTSDCLFPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 435
IRS1 XENTR STSGHGSTSDCMCPRRSSASVSGSPSDGGFISSDEYGSSPCDFRSSFRSV 442
∗∗∗∗∗∗∗∗∗∗∗ : ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

Figure 1: Continued.
Advances in Bioinformatics 5

IRS1 HUMAN TPDSLGHTPPARGEEELSNYICMGGKGPSTLTAPNGHYILSRGGNGHRCT 495

IRS1 CHLAE TPDSLGHTPPARGEEELSNYICMGGKGPSTLTAPNGHYILSRGGNGHRYT 495
IRS1 CHICK TPDSLGHTPPARGEEELSNYICMGGKGPSTLTAPNGHYILSRGANGHRCT 493
IRS1 MOUSE TPDSLGHTPPARGEEELSNYICMGGKGASTLAAPNGHYILSRGGNGHRYI 490
IRS1 RAT TPDSLGHTPPARGEEELSNYICMGGKGASTLTAPNGHYILSRGGNGHRYI 490
IRS1 CRIGR TPDSLGHTPPARGEEELSNYICMGGKGASTLTAPNGHYILSRGGNGHRYI 491
IRS1 PIG TPDSLGHTPPARGEEELSNYICMGGKGASTLAAPNGHYILPRGGNGHRCL 485
IRS1 XENTR TPDSLGHTPPAR-EEELNNYICMGKSGSHLQRSQQQRYQLSRGEE----- 486
∗∗∗∗∗∗∗∗∗∗∗∗ ∗∗∗∗ . ∗∗∗∗∗∗ . ∗ . : : : ∗ ∗ . ∗∗ :
IRS1 HUMAN PGTGLGTSPALAGDEAASAADLDNRFRKRTHSAGTSP-TITHQKTPSQSS 544
IRS1 CHLAE PGTGLGTSPALAGDEASSAADLDNRFRKRTHSAGTSP-TITHQKTPSQSS 544
IRS1 CHICK PGTGLGTSPALAGDEQTSAADLDNRFRKRTHSAGTSP-TITHQKTPSQSS 542
IRS1 MOUSE PGANLGTSPALPGDEAAGAADLDNRFRKRTHSAGTSP-TISHQKTPSQSS 539
IRS1 RAT PGATMGTSPALTGDEAAGAADLDNRFRKRTHSAGTSP-TISHQKTPSQSS 539
IRS1 CRIGR PGANLGTSPALTGDEATSAADLDNRFRKRTHSAGTSP-TISHQKTPSQSS 540
IRS1 PIG PGAGLGTSPALTGEEAGPASDLDNRFRKRTHSAGTSP-TISHQKTPSQSS 534
IRS1 XENTR ------------------HPDFDKVFRKRTHSSGTSPPTVSHQKTPSQS- 517
. ∗ : ∗ : ∗∗∗∗∗∗∗ : ∗∗∗∗ ∗ : : ∗∗∗∗∗∗∗∗
IRS1 HUMAN VASIEEYTEMMP-AYPPGGGSGGRLPGHRHSAFVPTRSYPEEGLEMHPLE 593
IRS1 CHLAE VASIEEYTEMMP-AYPPGGGSGGRLPGHRHSAFVPTHSYPEEGLEMHPLE 593
IRS1 CHICK VASIEEYTEMMP-AYPPGGGSGGRAWPGRLSAFVPTRSYPEEGLEMHPLE 591
IRS1 MOUSE VASIEEYTEMMPAAYPPGGGSGGRLPGYRHSAFVPTHSYPEEGLEMHHLE 589
IRS1 RAT VVSIEEYTEMMPAAYPPGGGSGGRLPGYRHSAFVPTHSYPEEGLEMHHLE 589
IRS1 CRIGR VASIEEYTEMMPAAYPPGGGSGGRLPSYRHSAFVPTHSYPEEGLEMHPLE 590
IRS1 PIG VASIEEYTEMMP-AYPPGGGSGGRMPNYRHSAFVPTHSYPEEGLEMHPLE 583
IRS1 XENTR --SIEEYTEMMP-------AHPVRLTSFRHSAFVPTYSYPEECLDLHLEG 558
∗∗∗∗∗∗∗∗∗∗ . ∗ ∗ ∗∗∗∗∗∗ ∗∗∗∗∗ ∗ : : ∗
IRS1 HUMAN RRGGHHRPDSSTLHTDDGYMPMSPGVAPVPSGRKGSGDYMPMSPKSVSAP 643
IRS1 CHLAE RRGGHHRPDSSTLHTDDGYMPMSPGVAPVPSSRKGSGDYMPMSPKSVSAP 643
IRS1 CHICK RRAGRTSPDSSTLHTDDGYMPMSPGWPPVSSGRKGNGDYMSMSPKSVSTP 641
IRS1 MOUSE RRGGHHRPDTSNLHTDDGYMPMSPGVAPVPSNRKGNGDYMPMSPKSVSAP 639
IRS1 RAT RRGGHHRPDSSNLHTDDGYMPMSPGVAPVPSNRKGNGDYMPMSPKSVSAP 639
IRS1 CRIGR RRGGHHRPDTSTLHTDDGYMPMSPGVAPVPSNRKGNGDYMPMSPKSVSAP 640
IRS1 PIG RRGGHHRQDTSSLHTDDGYMPMSPGVAPVPGTRKGSGDYMPMSPKSVSAP 633
IRS1 XENTR SRAN---------HTDDGYMPMSPGVAPVPTK---SNDYMPMSPKSVSAP 596
∗. . ∗∗∗∗∗∗∗∗∗∗∗∗ . ∗∗ . . . ∗∗∗ . ∗∗∗∗∗∗∗ : ∗
IRS1 HUMAN QQIINPIRRHPQRVDPNGYMMMSPSGGCSPDIGGGPSSSSSSSNAVPSGT 693
IRS1 CHLAE QQIINPIRRHPQRVDPNGYMMMSPSGGCSPDIGGGPSSSSSS--TVPSGS 691
IRS1 CHICK QQIINPITGPSERVYPNGYMMMSHSGGCSPDIGGGPSSSSSSSNAVPSGT 691
IRS1 MOUSE QQIINPIRRHPQRVDPNGYMMMSPSGSCSPDIGGG-SSSSSSISAAPSGS 688
IRS1 RAT QQIINPIRRHPQRVDPNGYMMMSPSGSCSPDIGGG-SCSSSSISAAPSGS 688
IRS1 CRIGR QQIINPIRRHPQRVDPNGYMMMSPSGSCSPDIGGG-SSSSS--GAAASGS 687
IRS1 PIG QQIINPIRRHPQRVDPNGYMMMSPSGSCSPDIGGGPSSGGSSSGAAPSGS 683
IRS1 XENTR QQIINPRRHS--AVDSNGYMMMSPSGSCS-----------------PDST 627
∗∗∗∗∗∗ ∗ . ∗∗∗∗∗∗∗ ∗ ∗ . ∗∗ . ..:
IRS1 HUMAN SYGKLWTNGVGGHHSHVLPHPKPPVESSGGKLLPCTGDYMNMSPVGDSNT 743
IRS1 CHLAE SYGKLWTKGVGAHNSQVLLHPKPPVESSGGKLLPCTGDYMNMSPVGDSNT 741
IRS1 CHICK SYGKLWTNGVGGHHSHVLPHPKPPVESSGGKLLPCTGDYMDMYPVGDSNT 741
IRS1 MOUSE SYGKPWTNGVGGHHTHALPHAKPPVESGGGKLLPCTGDYMNMSPVGDSNT 738
IRS1 RAT SYGKPWTNGVGGHHTHALPHAKPPVESGGGKLLPCTGDYMNMSPVGDSNT 738
IRS1 CRIGR SYGKPWTNGVGGHHSHALPHSKPPTESGSGKLLPCTGDYMNMSPVGDSNT 737
IRS1 PIG SYGKLWTNGVGGHHSHALPHPKLPVESSSGKLLSCTGDYMNMSPVGDSNT 733
IRS1 XENTR NYSKIWTNGTN---------PKLSIDSIEG-KLPCS-DYINMSPASGSTT 666
. ∗ . ∗ ∗∗ : ∗ . . . ∗ . : ∗ ∗ ∗ . ∗ : ∗∗ : : ∗ ∗ . . . ∗ . ∗
IRS1 HUMAN SSPSDCYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLST 793
IRS1 CHLAE SSPSDCYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLST 791
IRS1 CHICK SDPSLSYYGPEDPQHKPVLSYYSLPDAFKHPSGPGEPEEGARHQHLRLST 791
IRS1 MOUSE SSPSECYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLSS 788
IRS1 RAT SSPSECYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLSS 788
IRS1 CRIGR SSPSECYYGPEDPQHKPVLSYYSLPRSFKHTQRPGEPEEGARHQHLRLSS 787
IRS1 PIG SSPSDCYYGPEDPQHKPVLSYYSLPRSFKHTQRPGELEESARHQHLRLSS 783
IRS1 XENTR STPPDSYLNSIEESTKPVYSYFSLPRSFKHVHRKSE------DGNLRITA 710
∗ ∗ . . ∗ . . : . ∗∗∗ ∗∗ : ∗∗∗ : ∗∗∗ .∗ . : ∗∗ : : :
IRS1 HUMAN SSGRLLYAATADDSSSSTSSDSLGGGYCGARLEPSLPHPHHQVLQPHLPR 843
IRS1 CHLAE SSGRLLYAATADDSSSSTSSDSLGGGYCGARLEPSLPHPHHQVLQPHLPR 841
IRS1 CHICK SSGRLLYAATADDSSSSTSSDTLGGGYCGARLEPRLPHPHHQVLQPHLAR 841
IRS1 MOUSE SSGRLRYTATAEDSSSSTSSDSLGGGYCGARPESSLTHPHHHVLQPHLPR 838
IRS1 RAT SSGRLRYTATAEDSSSSTSSDSLGGGYCGARPESSVTHPHHHALQPHLPR 838
IRS1 CRIGR SSGRLLYAATAEDSSSSTSSDSLGGAYCGARPESGLPHPHHHVVQPHGPR 837
IRS1 PIG SSGRLLYAAAAEDSSSSTSSDSLGGGYCGARPEPGLPHLHHQVLQPHLPR 833
IRS1 XENTR NSGHNLYTE--DSSSSSTSSDSLGG------------------QDPQQPR 740
. ∗∗ : ∗ : : . ∗∗∗∗ ∗∗∗∗ : ∗∗∗ :∗ : . ∗
IRS1 HUMAN KVDT-AAQTNSRLARPTRLSLGDP-KASTLPRAREQQQQQQ--------- 882
IRS1 CHLAE KVDT-AAQTNSRLARPTRLSLGDP-KASTLPRAREQQQQQQQQQQQQQQQ 889
IRS1 CHICK KVDTSLAQTNSRLARPTRLSLGDP-KASTLPRAREQQQQSQ--------- 881
IRS1 MOUSE KVDT-AAQTNSRLARPTRLSLGDP-KASTLPRVREQQQQQQ--------- 877
IRS1 RAT KVDT-AAQTNSRLARPTRLSLGDP-KASTLPRVREQQQQQQQQQQ----- 881
IRS1 CRIGR KVDT-AAQTNRRLPRPTRLSLGDP-KASTFPRVWEQQQ------------ 873
IRS1 PIG KVDT-AAQTNNRLARPTRLSLGDP-KASTLPRAREQPPQP---------- 871
IRS1 XENTR KGET--CIQGKRLTRPTRLSLENSSKASTLPRAREPAL------------ 776
∗ : ∗ . . ∗∗ . ∗∗∗∗∗∗∗ : . ∗∗∗∗ : ∗∗ . ∗

Figure 1: Continued.
6 Advances in Bioinformatics

IRS1 HUMAN --PLLHPPEPKSPGEYVNIEFGSDQSGYLSGPVAFHSSPSVRCPSQLQPA 930

IRS1 CHLAE QQPLLHPPEPKSPGEYVNIEFGSDQPGYLSGPVASRSSPSVRCPSQLQPA 939
IRS1 CHICK --PFACPPEPKSPGEYVNIEFGSDQSGYLSGPVAFHSSPSVRCPSQLQPA 929
IRS1 MOUSE --SSLHPPEPKSPGEYVNIEFGSGQPGYLAGPATSRSSPSVRCPPQLHPA 925
IRS1 RAT --SSLHPPEPKSPGEYVNIEFGSGQPGYLAGPATSRSSPSVRCLPQLHPA 929
IRS1 CRIGR --PSLHPPEPKSPGEYVNIEFGSGQPGYLAGPATSHSSPSVRCPPQLHPA 921
IRS1 PIG --ALLHPPEPKSPGEYVNIEFGGDQPGYLSGPVASHSAPSVRCSSQLQPA 919
IRS1 XENTR ------PPEPKSPGEYVNIEFN---DKVFSGGLMPSMCPLPFVQSRVVP- 816
∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . ::∗ .∗ .:: ∗
IRS1 HUMAN PREEETGTEEYMKMDLGPGRRAAWQESTGVE------MGRLGPAPPGAAS 974
IRS1 CHLAE PREEETGTEEYMKMDLGPGRRAAWQESTGVE------MGRLGPAPPGAAS 983
IRS1 CHICK PREEETGTEEYMKMDLGPGRRAAWQERTGVE------MGRLGPAPPGAAS 973
IRS1 MOUSE PREE-TGSEEYMNMDLGPGRRATWQESGGVE------LGRIGPAPPGSAT 968
IRS1 RAT PREE-TGSEEYMNMDLGPGRRATWQESGGVE------LGRVGPAPPGAAS 972
IRS1 CRIGR PREE-TGSEEYMNMDLGPGRRATWQESGGVE------LGRVGPAPPGAAT 964
IRS1 PIG PREENTGAEEYMNMDLGPGRRAIWQESAGVQ------PGRVGPAPPGAAS 963
IRS1 XENTR QREN---LSEYMNMDLGVWRAKTSYASTSSSSYEPPYKPVSSVCPTETCS 863
∗∗ : . ∗∗∗ : ∗∗∗∗ ∗ . . . .∗. : . :
IRS1 HUMAN ICRPTRAVPSSRGDYMTMQMSCPRQSYVDTSP----AAPVSYADMRTGIA 1020
IRS1 CHLAE ICRPTRAVPSSRGDYMTMQMSCPRQSYVDTSP----IAPVSYADMRTGIA 1029
IRS1 CHICK ICRPT-AVPSSRGDYMTMQMSCPRQSYVDTSP----AAPVSYADMRTGIA 1018
IRS1 MOUSE VCRPTRSVPNSRGDYMTMQIGCPRQSYVDTSP----VAPVSYADMRTGIA 1014
IRS1 RAT ICRPTRSVPNSRGDYMTMQIGCPRQSYVDTSP----VAPVSYADMRTGIA 1018
IRS1 CRIGR ICRPTRSVPGSRGDYMTMQIGCPRQSYVDTSP----VAPVSYADMRTSIG 1010
IRS1 PIG VCRPTRAVPSSRGDYMTMQMGCPRQSYVDTSP----VAPISYADMRTGIV 1009
IRS1 XENTR SSRPPIRGKTNSRDYMSMQLSALCSDYSQVPPTRITAKPITLSSNKSNYA 913
. ∗∗ . . ∗∗∗ : ∗∗ : . . . . ∗ : . . ∗ ∗: : : . :: .
IRS1 HUMAN AEEVSLPRATMAAASSSSAASAS--PTGPQGAAELAAHSSLLGGPQGPGG 1068
IRS1 CHLAE AEEVSLPRATMAAAASSSAASAS--PTGPQGAAELAAHSSLLGGAQGPGG 1077
IRS1 CHICK AEEVSLPRATIAAASSSSTTSAS--DWARQ-APELAAHFVLLGGPQGPGG 1065
IRS1 MOUSE AEKASLPRPTGAAPPPSSTASS--SVTPQGATAEQATHSSLLGGPQGPGG 1062
IRS1 RAT AEKVSLPRTTGAAPPPSSTASASASVTPQGA-AEQAAHSSLLGGPQGPGG 1067
IRS1 CRIGR AEKVSLPRTTGAAPSSSSTASAS-PAAPEGA-AEQAAHSSLLGGPQGPGG 1058
IRS1 PIG VEEASLPGATAAAPSASSATSAS-SAAPQGA-GELAACSSLLGGPQGPGG 1057
IRS1 XENTR EMSSGGVSDNIPAIPQTSNSSLS-----------EASRSSLLG--QG-SG 949
. . . . ∗ . :∗ : ∗ ∗: ∗∗∗ ∗∗ . ∗
IRS1 HUMAN MS-AFTRVNLSPNRNQSAKVIRADPQGCRRRHSSETFSSTPSATRVGNTV 1117
IRS1 CHLAE MS-AFTRVNLSPNRNQSAKVIRADPQGCRRRHSSETFSSTPSATRVGNTV 1126
IRS1 CHICK MSGSFTRANLSPNRNQSAKVIRADTQGCRRIASSQTLSSTPSSTRWHTHV 1115
IRS1 MOUSE MS-AFTRVNLSPNHNQSAKVIRADTQGCRRRHSSETFSAP---TRAGNTV 1108
IRS1 RAT MS-AFTRVNLSPNHNQSAKVIRADTQGCRRRHSSETFSAP---TRAANTV 1113
IRS1 CRIGR MS-AFTRVNLSPNHNQSAKVIRADTQGCRRRHSSETFSAP---TRAGNTV 1104
IRS1 PIG LS-AFTRVNLSPNRNQSAKVIRADPQGCRRRHSSETFSSTPSATRAGNTV 1106
IRS1 XENTR PS-AFTRVSLSPNRNQSAKVIRAGDPQGRRRHSSETFSSTPTTARVTS-- 996
∗ : ∗∗∗ . .∗∗∗∗ : ∗∗∗∗∗∗∗∗∗ . ∗∗ ∗∗ : ∗ : ∗ : . :∗ .
IRS1 HUMAN PFGAGAAVGGGG----GSSSSSEDVKRHSSASFENVWLRPGELGGAPKEP 1163
IRS1 CHLAE PFGAGAAIGGSG----GSSSSSEDVKRHSSASFENVWLRPGELGGAPKEP 1172
IRS1 CHICK PLRAGAAVGGIR----VSASSEQDVKPHSSASFENVWLRPGELGGAPKEP 1161
IRS1 MOUSE PFGAGAAVGGSGGGGGGGS---EDVKRHSSASFENVWLRPGDLGGVSKES 1155
IRS1 RAT SFGAGAAGGGSG----GGS---EDVKRHSSASFENVWLRPGDLGGASKES 1156
IRS1 CRIGR SFGAGAAVGGSGGGAGGGSSSSEDVKRHSSASFENVWLRPGDLGGASKET 1154
IRS1 PIG PFGGGAAVGGGG----GGSSSTEDVKRHSSASFENVWLRPGELGGAPKEL 1152
IRS1 XENTR -----------------GPVSGEDVKRHSSASFENVWLKPGEIARRDS-- 1027
.. : ∗∗∗ ∗∗∗∗∗∗∗∗∗∗∗ : ∗∗ : : . .
IRS1 HUMAN AKLCGAAGGLENGLNYIDLDLVKDFKQCPQECTPEPQPPPPPPPHQPLGS 1213
IRS1 CHLAE AQLCGAAGGLENGLNYIDLDLVKDFKQRPQECTPQPQPPPPPPPHQPLGS 1222
IRS1 CHICK AKLCGAAGGLENGLNYIDLDLVKDFKQCPQECTPEPQPPPPPPPHQPLGS 1211
IRS1 MOUSE APVCGAAGGLEKSLNYIDLDLAK---ERSQDCPSQQQSLPPPPPHQPLGS 1202
IRS1 RAT APGCGAAGGLEKSLNYIDLDLVKDVKQHPQDCPSQQQSLPPPPPHQPLGS 1206
IRS1 CRIGR AQVCGAAGGLESNLNYIDLDLAKDVRQRPQECPSQQQPLPPPAPHQPSRN 1204
IRS1 PIG AQMCGAAGGLENGLNYIDLDLVKDFKQRPQERPPQPQPPPPPPPHQPLGN 1202
IRS1 XENTR ---LQPSDHTHNGLNYIDLDLAKDLSGLDHCN------------------ 1056
. : . . . . ∗∗∗∗∗∗∗∗ . ∗ :
IRS1 HUMAN GESSSTRRSSEDLSAYASISFQKQPEDRQ 1242
IRS1 CHLAE SESSSTRRSSEDLSAYASISFQKQPEDLQ 1251
IRS1 CHICK GESSSTRRSSEDLSAYASTSFQKQPEDRQ 1240
IRS1 MOUSE NEGNSPRRSSEDLSNYASISFQKQPEDRQ 1231
IRS1 RAT NEGSSPRRSSEDLSTYASINFQKQPEDRQ 1235
IRS1 CRIGR NEGSSPRRSSEDLSTYASINFQKQPEDHQ 1233
IRS1 PIG SESSSTSRSSEDLSAYASIRFQKQPED-- 1229
IRS1 XENTR SHQSGVSHPSDDLSTYASITFHKLEEHRN 1085
. . . . : . ∗ : ∗∗∗ ∗∗∗ ∗ : ∗ ∗ .

Figure 1: Multiple alignments of eight sequences (human, green monkey, pig, chicken, Chinese hamster, mouse, rat, and western clawed frog).
ClustalW was used to align these different sequences. The conserved sequence (highlighted red) is marked by an asterisk, semiconserved
(highlighted yellow) substitution by a single dot, and conserved substitution (highlighted green) by a double dot. Multiple alignment showed
that Ser-11, 24, 36, 57, 63, 68, 193, 199, 217, 228, 243, 261, 270, 272, 273, 274, 277, 281, 295, 307, 312, 323, 329, 330, 337, 345, 348, 350, 362, 372, 374,
383, 385, 388, 391, 393, 394, 395, 396, 398, 402, 404, 412, 413, 415, 417, 419, 421, 427, 428, 433, 434, 440, 441, 444, 449, 527, 531, 541, 543, 547,
574, 581, 616, 636, 639, 641, 666, 668, 672, 720, 741, 744, 763, 766, 795, 807, 808, 809, 810, 812, 813, 862, 869, 892, 1038, 1041, 1070, 1078, 1084,
1100, 1101, 1105, 1142, 1143, 1145, 1223, 1227, and 1231; Thr-88, 188, 191, 231, 252, 305, 309, 311, 335, 387, 397, 403, 446, 453, 525, 530, 533, 539, 552,
579, 608, 700, 743, 811, 847, 859, 870, 1073, and 1103; and Tyr-18, 46, 47, 87, 107, 183, 431, 465, 483, 551, 582, 612, 632, 662, 695, 732, 750, 764,
800, 896, 941, 989, 1001, 1179, and 1229 are highly conserved residues. Meanwhile, Ser-137, 268, 303, 380, 770, 792, 815, 910, 974, 1011, 1037, and
1106; Thr-351, 354, 535, 693, 729, 793, 991, 1017, and 1111; and Tyr-765 showed conserved substitutions. Ser-58, 78, 99, 105, 135, 139, 189, 315, 341,
463, 486, 629, 691, 694, 747, 794, 918, 925, 985, 995, 1000, 1005, 1025, 1035, 1131, 1132, 1217, 1218, and 1222; and Thr-300, 979, 1004, 1030, and 1107
showed semiconserved substitutions.
Advances in Bioinformatics 7

IRS2 HUMAN MASPPRHGPPGPASGDGPNLNNNNNNNN-HSVRKCGYLRKQKHGHKRFFV 49

IRS2 RAT MASAPLPGPPASAGGDGPNLNNNNNNNNNHSVRKCGYLRKQKHGHKRFFV 50
IRS2 MOUSE MASAPLPGPPASAGGDGPNLNNNNNNNN-HSVRKCGYLRKQKHGHKRFFV 49
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN LRGPGAGGDEATAGGGSAPQPPRLEYYESEKKWRSKAGAPKRVIALDCCL 99
IRS2 RAT LRGPGTGGEEAAAAGGSPPQPPRLEYYESEKKWKSKAGAPKRVIALDCCL 100
IRS2 MOUSE LRGPGTGGDEASAAGGSPPQPPRLEYYESEKKWRSKAGAPKRVIALDCCL 99
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN NINKRADAKHKYLIALYTKDEYFAVAAENEQEQEGWYRALTDLVSEGRAA 149
IRS2 RAT NINKRADAKHKYLIALYTKDEYFAVAAENEQEQEGWYRALTDLVSEGRSG 150
IRS2 MOUSE NINKRADAKHKYLIALYTKDEYFAVAAENEQEQEGWYRALTDLVSEGRSG 149
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN AGDAPPAAAPAASCSASLPGALGGSAGAAGAEDSYGLVAPATAAYREVWQ 199
IRS2 RAT DGGSGTTGG---SCSASLPGALGGSAGAAGCDDNYGLVTPATAVYREVWQ 197
IRS2 MOUSE EGGSGTTGG---SCSASLPGVLGGSAGAAGCDDNYGLVTPATAVYREVWQ 196
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN VNLKPKGLGQSKNLTGVYRLCLSARTIGFVKLNCEQPSVTLQLMNIRRCG 249
IRS2 RAT VNLKPKGLGQSKNLTGVYRLCLSARTIGFVKLNCEQPSVTLQLMNIRRCG 247
IRS2 MOUSE VNLKPKGLGQSKNLTGVYRLCLSARTIGFVKLNCEQPSVTLQLMNIRRCG 246
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN HSDSFFFIEVGRSAVTGPGELWMQADDSVVAQNIHETILEAMKALKELFE 299
IRS2 RAT HSDSFFFIEVGRSAVTGPGELWMQADDSVVAQNIHETILEAMKALKELFE 297
IRS2 MOUSE HSDSFFFIEVGRSAVTGPGELWMQADDSVVAQNIHETILEAMKALKELFE 296
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN FRPRSKSQSSGSSATHPISVPGARRHHHLVNLPPSQTGLVRRSRTDSLAA 349
IRS2 RAT FRPRSKSQSSGSSATHPISVPGARRHHHLVNLPPSQTGLVRRSRTDSLAA 347
IRS2 MOUSE FRPRSKSQSSGSSATHPISVPGARRHHHLVNLPPSQTGLVRRSRTDSLAA 346
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN TPPAAKCSSCRVRTASEGDGGAAAGAAAAGARPVSVAGSPLSPGPVRAPL 399
IRS2 RAT TPPAAKCTSCRVRTASEGDGGAAGGAGTAGGRPMSVAGSPLSPGPVRAPL 397
IRS2 MOUSE TPPAAKCTSCRVRTASEGDGGAAGGAGTAGGRPMSVAGSPLSPGPVRAPL 396
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN SRSHTLSGGCGGRGSKVALLPAGGALQHSRSMSMPVAHSPPAATSPGSLS 449
IRS2 RAT SRSHTLSAGCGGRPSKVALAPAGGALQHSRSMSMPVAHSPPAATSPGSLS 447
IRS2 MOUSE SRSHTLSAGCGGRPSKVTLAPAGGALQHSRSMSMPVAHSPPAATSPGSLS 446
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN SSSGHGSGSYPPPPGPHPPLPHPLHHGPGQRPSSGSASASGSPSDPGFMS 499
IRS2 RAT SSSGHGSGSYPLPPGSHPHLPHPLHHPQCQRPSSGSASASGSPSDPGFMS 497
IRS2 MOUSE SSSGHGSGSYPLPPGSHPHLPHPLHHPQGQRPSSGSASASGSPSDPGFMS 496
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN LDEYGSSPGDLRAFCSHRSNTPESIAETPPARDGGGGGEFYGYMTMDRPL 549
IRS2 RAT LDEYGSSPGDLRAFSSHRSNTPESIAETPPARDGSGG-ELYGYMSMDRPL 546
IRS2 MOUSE LDEYGSSPGDLRAFSSHRSNTPESIAETPPARDGSGG-ELYGYMSMDRPL 545
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN SHCGRSYRRVSGDAAQDLDRGLRKRTYSLTTPARQRPVPQPSSASLDEYT 599
IRS2 RAT SHCGRPYRRVSGDGAQDLDRGLRKRTYSLTTPARQRQVPQPSSASLDEYT 596
IRS2 MOUSE SHCGRPYRRVSGDGAQDLDRGLRKRTYSLTTPARQRQVPQPSSASLDEYT 595
IRS2 CRIGR --------------------------------------------------
---------
IRS2 HUMAN LMRATFSGSAGRLCPSCPASSPKVAYHPYPEDYGDIEIGSHRSSSSNLGA 649
IRS2 RAT LMRATFSGSSGRLCPSLPASSPKVAYNPYPEDYGDIEIGSHKSSSSNLGA 646
IRS2 MOUSE LMRATFSGSSGRLCPSFPASSPKVAYNPYPEDYGDIEIGSHKSSSSNLGA 645
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN DDGYMPMTPGAALAGSGSGSCRSDDYMPMSPASVSAPKQILQPRAAAAAA 699
IRS2 RAT DDGYMPMTPGAALRSGGPNSCKSDDYMPMSPTSVSAPKQILQP----RSA 692
IRS2 MOUSE DDGYMPMTPGAALRSGGPNSCKSDDYMPMSPTSVSAPKQILQP----RLA 691
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN AAVPSAGPAGPAPTSAAGRTFPASGGGYKASSPAESSPEDSGYMRMWCGS 749
IRS2 RAT AALPPSGAAVPAPPSGAGRTFPVNGGGYKASSPAESSPEDSGYMRMWCGS 742
IRS2 MOUSE AALPPSGAAVPAPPSGVGRTFPVNGGGYKASSPAESSPEDSGYMRMWCGS 741
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN KLSMEHADGKLLPNGDYLNVSPSDAVTTGTPPDFFSAALHPGGEPLRGVP 799
IRS2 RAT KLSMENPDPKLLPNGDYLNMSPSEAGTAGTPPDFFSAALRPGGEALKGVP 792
IRS2 MOUSE KLSMENPDPKLLPNGDYLNMSPSEAGTAGTPPD-FSAALRGGSEGLKGIP 790
IRS2 CRIGR --------------------------------------------------
IRS2 HUMAN GCCYSSLPRSYKAPYTCGG-DSDQYVLMSSPVGRILEEERLEPQATPGPS 848
IRS2 RAT GHCYSSLPRSYKAPCTCGGGDNDQYVLMSSPVGRILEEERLEPQATPG-A 841
IRS2 MOUSE GHCYSSLPRSYKAPCSCSG-DNDQYVLMSSPVGRILEEERLEPQATPG-A 838
IRS2 CRIGR --------------------------------------------------

Figure 2: Continued.
8 Advances in Bioinformatics

IRS2 HUMAN QAASAFGAGPTQPPHPVVPSPVRPSG--GRPEGFLGQRGRAVRPTRLSLE 896

IRS2 RAT GTFGAAGGSHTQPHHSAVPSSMRPSGIVGRPEGFLGQRCRAVRPTRLSLE 891
IRS2 MOUSE GTFGAAGGSHTQPHHSAVPSSMRPSAIGGRPEGFLGQRCRAVRPTRLSLE 888
IRS2 CRIGR ---------------------MRPSGSGGRPEGFLGQRCRAVRPTRLSLE 29
: ∗∗∗ . ∗∗∗∗∗∗∗∗∗∗ ∗∗∗∗∗∗∗∗∗∗∗
IRS2 HUMAN GLPSLPSMHEYPLPPEPKSPGEYINIDFGEPGARLSPPAPPLLASAASSS 946
IRS2 RAT GLQTLPSMQEYPLPTEPKSPGEYINIDFGEGGTRLSPPAPPLLASAASSS 941
IRS2 MOUSE GLQTLPSMQEYPLPTEPKSPGEYINIDFGEAGTRLSPPAPPLLASAASSS 938
IRS2 CRIGR GLQTLPSMQEYPLPAEPKSPGEYINIDFGEAGTRLSPPAPPLLASAASSS 79
∗∗ : ∗∗∗ ∗ : ∗∗∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ ∗ : ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗
IRS2 HUMAN SLLSASSPASSLGSGTPGTSSDSRQRSPLSDYMNLDFSSPKSPKPGAPSG 996
IRS2 RAT SLLSASSPASSLGSGTPGTSSDSRQRSPLSDYMNLDFSSPKSPKPSTRSG 991
IRS2 MOUSE SLLSASSPASSLGSGTPGTSSDSRQRSPLSDYMNLDFSSPKSPKPSTRSG 988
IRS2 CRIGR SLLSASSPASSLGSGTPGTSSDSRQRSPLSDYMNLDFSSPKSPKPGTHSG 129
∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . : ∗∗
IRS2 HUMAN HPVGSLDGLLSPEASSPYPPLPPRPSASPSSSLQPPPPPPAPGELYRLPP 1046
IRS2 RAT DTVGSIDGLLSPEASSPYPPLPPRPSASPSSLQQPLPP--APGDLYRLPP 1039
IRS2 MOUSE DTVGSMDGLLSPEASSPYPPLPPRPSTSPSSLQQPLPP--APGDLYRLPP 1036
IRS2 CRIGR DTVGSMDGLLSPEASSPYPPLPPRPSASPSSLQQPLPP--APGELYRLPP 177
. . ∗∗∗ : ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ : ∗∗∗∗ ∗∗ ∗∗ ∗∗∗ : ∗∗∗∗∗∗
IRS2 HUMAN ASAVATAQGPGAASSLSSDTGDNGDYTEMAFGVAATPPQPIAAPPKPEAA 1096
IRS2 RAT AT-AATSQGPTAGSSMSSEPGDNGDYTEMAFGVAATPPQPIAAPPKPEGA 1088
IRS2 MOUSE AS-AATSQGPTAGSSMSSEPGDNGDYTEMAFGVAATPPQPIVAPPKPEGA 1085
IRS2 CRIGR AS-AATSQGPTAGSSKSSEPGDNGDYTEMAFGVAAPPPQPIAAPPKPEGA 226
∗ : . ∗∗ : ∗∗∗ ∗ . ∗∗ ∗∗ : . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . ∗∗∗∗∗ . ∗∗∗∗∗∗ . ∗
IRS2 HUMAN RVASPTSGVKRLSLMEQVSGVEAFLQASQPPDPHRGAKVIRADPQGGRRR 1146
IRS2 RAT RVTSPTSGLKRLSLMDQVSGVEAFLQVSQPPDPHRGAKVIRADPQGGRRR 1138
IRS2 MOUSE RVASPTSGLKRLSLMDQVSGVEAFLQVSQPPDPHRGAKVIRADPQGGRRR 1135
IRS2 CRIGR RVTSPTSGLKRLSLMEQVSGVEAFLQVSQPPDPHRGAKVIRADPQGGRRR 276
∗∗ : ∗∗∗∗∗ : ∗∗∗∗∗∗ : ∗∗∗∗∗∗∗∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗
IRS2 HUMAN HSSETFSSTTTVTPVSPSFAHNPKRHNSASVENVSLRKSSEGGVGVGPGG 1196
IRS2 RAT HSSETFSSTTTVTPVSPSFAHNSKRHNSASVENVSLRKSSEGNSILG--G 1186
IRS2 MOUSE HSSETFSSTTTVTPVSPSFAHNSKRHNSASVENVSLRKSSEGSSTLG--G 1183
IRS2 CRIGR HSSETFSSTTTVTPVSPSFAHNSKRHNSASVENVSLRKSSEGSGILG--G 324
∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ . : ∗ ∗
IRS2 HUMAN GDEPPTSPRQLQP--APPLAPQGRPWTPGQPGGLVGCPGSGGSPMRRETS 1244
IRS2 RAT SDEPSTSPGQAQPSAGVPPAPQARPWNPGQPGALIGCPGGSSSPMRRETS 1236
IRS2 MOUSE GDEPPTSPGQAQPLVAVPPVPQARPWNPGQPGALIGCPGGSSSPMRRETS 1233
IRS2 CRIGR SDEPPTSPGQAQPSVAVPPAPQARPWNPGQPGALIGCPGGSSSPMRRETS 374
. ∗∗∗ . ∗∗∗ ∗ ∗∗ . ∗ . ∗ ∗ . ∗∗∗ . ∗∗∗∗∗ . ∗ : ∗∗∗∗ . . . ∗∗∗∗∗∗∗∗
IRS2 HUMAN AGFQNGLNYIAIDVREEPGLPPQPQPPPPPLPQPGDKSSWGRTRSLGGLI 1294
IRS2 RAT VGFQNGLNYIAIDVRGEQGSLAQSQPQH---PQPGDKNSWGRTRSLGGLL 1283
IRS2 MOUSE VGFQNGLNYIAIDVRGEQGSLAQSQ------PQPGDKNSWSRTRSLGGLL 1277
IRS2 CRIGR VGFQNGLNYIAIDVRDEQGSLSQTQPQH---PQPGDKSSWGRTRSLGGLL 421
. ∗∗∗∗∗∗∗∗∗∗∗∗∗∗ ∗ ∗ . ∗ . ∗ ∗∗∗∗∗∗. ∗∗ . ∗∗∗∗∗∗∗∗ :
IRS2 HUMAN SAVGVGSTGGGCGGPGPGALPPANTYASIDFLSHHLKEATIVKE 1338
IRS2 RAT GTVGGSGTSGVCGGPGTGALPSASTYASIDFLSHHLKEATVVKG 1327
IRS2 MOUSE GTVGGSGASGVCGGPGTGALPSASTYASIDFLSHHLKEATVVKE 1321
IRS2 CRIGR GTVGSTGSSGVCGGPGTGALPSASTYASIDFLSHHLKEATVVKE 465
. : ∗∗ . : . ∗ ∗∗∗∗∗ . ∗∗∗∗ .∗ . ∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗ : ∗∗

Figure 2: Multiple alignments of three sequences (human, rat, and mouse). ClustalW was used to align these different sequences. The
conserved sequence (highlighted red) is marked by an asterisk, semiconserved (highlighted yellow) substitution by a single dot, and conserved
substitution (highlighted green) by a double dot. Ser-3, 30, 66, 78, 84, 144, 162, 164, 166, 174, 210, 222, 237, 251, 253, 262, 277, 304, 306, 308,
309, 311, 312, 318, 334, 342, 346, 358, 365, 384, 388, 391, 400, 402, 406, 414, 428, 430, 432, 438, 444, 447, 449, 450, 451, 452, 456, 458, 482, 483,
485, 487, 489, 491, 493, 499, 505, 506, 515, 518, 523, 550, 560, 577, 591, 592, 594, 606, 608, 615, 619, 620, 639, 642, 643, 644, 645, 669, 672, 679,
682, 684,, 714, 730, 731, 735, 736, 740, 749, 752, 770, 772, 785, 804, 805, 809, 827, 828, 868, 873, 894, 903, 915, 932, 941, 944, 945, 946, 947, 950,
952, 953, 956, 957, 960, 966, 967, 969, 973, 976, 984, 985, 988, 995, 1001, 1007, 1011, 1012, 1022, 1024, 1026, 1027, 1060, 1061, 1063, 1064, 1100, 1103,
1109, 1115, 1124, 1148, 1149, 1153, 1154, 1162, 1164, 1174, 1176, 1181, 1185, 1186, 1203, 1237, 1244, 1283, 1289, 1322 and 1327; Thr-117, 140, 191, 214, 225,
239, 265, 286, 314, 336, 344, 350, 363, 404, 443, 520, 527, 575, 579, 580, 599, 604, 657, 719, 776, 779, 844, 859, 891, 962, 965, 1052, 1073, 1082,
1102, 1151, 1155, 1156, 1157, 1159, 1202, 1243, 1287, 1319, and 1334 and Tyr-36, 75, 76, 111, 116, 121, 136, 184, 194, 217, 459, 503, 540, 542, 556, 576, 598,
625, 628, 632, 653, 675, 727, 742, 766, 803, 810, 823, 907, 919, 978, 1014, 1042, 1072, 1253, and 1320 are highly conserved residues. Meanwhile,
Ser-357, 848, 900, and 1048 and Thr-61, 544, 777, 815, and 1302 showed conserved substitutions. Ser-14, 183, 555, 665, 667, 704, 723, 820, 852,
1234, 1282, 1295, and 1301 and Thr-713, 1066, and 1221 showed semiconserved substitutions.

159 (89.8%) were conserved, 4 (2.25%) were conserved substi-
tutions, and 13 (7.34%) were semiconserved. While for total
53 Thr residues, 45 (84.9%) were conserved, 5 (9.4%) were
conserved substitutes, and 3 (5.6%) were semiconserved. Tyr
residues showed maximum conservation status. Out of 37 Tyr
residues, 36 (97.29%) were conserved.
3.3. Predicted 3D Structures of Human IRS-1 and IRS-2
Proteins. 3D structure of protein plays important role in
determining the exact and precise functions of any protein.
However, the whole 3D structures of IRS-1 and IRS-2 proteins
have not been determined yet. To assess this difficulty we
predict the 3D structure of these proteins by homology and
Advances in Bioinformatics
ab initio modeling. We used many servers to predict the
whole structure of both proteins and I-TASSER was the only
server that predicts the complete structure of these pro-
teins. I-TASSER predicted five models for each 3D structure
with minimum 𝑧-score. The energy of these structures was
minimized using YASARA software. To determine whether
these structures truly represent or are nearer to possible
future true structures, their geometry had been accessed by
drawing Ramachandran plots that represent most reliable and
suitable source to check predicted 3D structure’s geometry.
The Ramachandran plot analysis is a method to determine
the phi and psi torsion angles between amino acids and
compare this information to experimentally verified data
as well as standard geometrical properties of protein 3D
structures. Five models each were predicted by I-TASSER
for constructing Ramachandran plots assessing both IRS-
1 and IRS-2. Figures 7(a) and 7(b) represent the predicted
3D structures, Ramachandran plot analysis, and geometrical
properties of IRS-1 and IRS-2.
On the basis of Ramachandran plot analysis we proposed
predicted model #2 (Figure 3(a)) as possible 3D structure
for IRS-1 protein. In this structure 94.7% residues were
in Ramachandran allowed region with only 5.32% outliers.
No residues with bad bonds were found while only 1.06%
residues with bad angles were predicted. However, the c𝛽
deviations for these predicted models were not predicted by
server.
Ramachandran plot analysis was also performed for
predicted structures for IRS-2 protein as shown in Figure 3(b)
and model #1 was selected as best predicted model. The
percentage of compliance for the predicted structure of IRS-
2 with the Ramachandran plot indicates that 82.2% (361)
of residues reside in the favored region while 94.3% (414)
of all residues exist within the allowed region with only
9 C𝛽 deviations > 0.25A. Only 5.69% of residues exist
within the disallowed region of the plot. The 3D structures
of IRS-1 and IRS-2 proteins predicted by I-TASSER were
submitted to online database PMDB—Protein Model Data-
base (http://bioinformatics.cineca.it/PMDB/). PMDB acces-
sion numbers are given in front of each respective model in
Figures 3(a) and 3(b).
IRS-1 and IRS-2 predicted 3D structures had been com-
pared with each other by FATCAT and visualized by using
Chimera as shown in Figure 4. IRS-1 structure was shown in
red while IRS-2 in green. Both structures were significantly
different (𝑃 value 9.01 E-01; Raw score = 251.71). The structure
alignment has 546 equivalent positions with an RMSD of
11.03, with five twists. Both structures were found to have
identity 4.02% and similarity 8.71%. This information leads
to the conclusion that both IRS-1 and IRS-2 proteins regulate
insulin singling in different ways.
3.4. Posttranslational Modification Prediction and Analysis
3.4.1. Experimentally Verified Phosphorylation Sites on IRS-1
and IRS-2. Phospho.ELM is the website that contains data
about experimentally verified phosphorylated residues and
kinases involved in proteins to ease research work. Human
IRS-1 has more than 40 Ser, Thr, and Tyr residues that were
9
found to be phosphorylated experimentally. These include
Ser-24, 268, 270, 272, 274, 307, 312, 323, 341, 345, 348, 374,
527, 531, 574, 616, 629, 636, 639, 794, 892, 1078, 1101, 1145, 1222,
and 1223 Thr-530, and 1111, Tyr-46, 612, 662, and 896. Uniprot
database also showed phosphorylation potential on Ser-3, 99,
329, 1223, and Tyr-465, 989, and 1229 on basis of similarity.
Different kinases and groups of kinases like CK2, PLK1, IKK,
PKC, MAPK3, SIK, AMPK, INSR, and RSK1 were involved in
phosphorylation of IRS-1 on same or different residues.
For human IRS-2 protein phosphorylation had been
verified on Ser-304, 306, 312, 334, 346, 365, 384, 388, 391,
518, 523, 550, 560, 577, 594, 608, 620, 679, 714, 730, 731, 735,
736, 770, 772, 828, 894, 915, 973, 1100, 1103, 1109, 1148, 1149,
1162, 1174, 1176, 1203, and 1283, Thr-350, 363, 520, 527, 713,
777, 779, 1151, 1159, and 1202, and Tyr-75, 184, 653, 675, 823,
and 919 residues. While IKK kinase group was involved in
phosphorylation, Uniprot database showed phosphorylation
on four more residues including above, that is, Thr-527, 579,
580 and Tyr-1253.
3.4.2. Predicted Phosphorylation Sites in IRS-1. To predict
phosphorylated residues in human IRS-1, NetPhos was used.
Figure 5(a) is a graphical representation of phosphorylation
potential of the Ser, Thr, and Tyr residues in IRS-1. NetPhos
predicted phosphorylation on 113 Ser, 13 Thr, and 20 Tyr
residues including all experimentally verified phosphoryla-
tion sites except Ser-323, 374, 794 and Thr-530 and 1111 as
shown in Table S1 available online at http://dx.doi.org/10.1155/
2014/324753.
3.4.3. Predicted Phosphorylation Sites in IRS-2. Similar to
IRS-1, web server NetPhos 2.0 also made prediction of
phosphorylated residues in human IRS-2. Figure 5(b) is a
graphical representation of phosphorylation potential of the
Ser, Thr, and Tyr residues in IRS-2. These predicted phospho-
rylation residues include all experimentally confirmed sites
except Ser-312, 388, 518, 679, 714, 722, 730, 772, 828, 1103, 1179,
Thr-350, 527, 579, 713, 1151, 1202, and Tyr-75 as shown in Table
S2.
3.4.4. Predicted and Experimentally Verified Kinases in IRS-1.
Various kinases involved in phosphorylation of human IRS-
1. NetPhosK 1.0, Scansite, and KinasePhos 2.0 were used to
assess possible kinases on IRS-1. The results obtained from
these three servers had shown the involvement of various
kinases in phosphorylation of human IRS-1. GSK3, PKC,
and PKA showed maximum involvement in phosphorylation
while other kinases like PKG, p38MAPK, cdk5, CKII, cdc2,
ATM, and CKI also showed potential for phosphorylation
of many Ser and Thr residues. INSR, syk, SRC, and insulin
receptor kinase are the most predicted kinases for tyrosine
phosphorylation as shown in Table 1S.
3.4.5. Predicted and Experimentally Verified Kinases in IRS-
2. Similar to IRS-1, NetPhosK 1.0, Scansite, and KinasePhos
2.0 predicted many kinases involved in phosphorylation
of human IRS-2. GSK3, PKC, PKA, 14-3-3 Mode 1, PKG,
p38MAPK, cdk5, CKII, cdc2, ATM, and CKI are the most
10 Advances in Bioinformatics

Human IRS-1 predicted Ramachandaran plots of Geometrical analysis of

structures by I-TASSER predicted structures predicted structures
180
PMDB ID: PM0079784
Poor rotamers = 48 (4.37%)
Ramachandran outliers = 102 (8.23%)
Ψ 0 Ramachandran favoured = 987 (79.6%)
Ramachandran allowed = 91.8%
C𝛽 deviations > 0.25A = N.A
Residues with bad bonds = 2/4967 (0.04%)
Predicted model # 1 −180 Residues with bad angles = 50/6207 (0.81%)
−180 0 180
Φ
180
PMDB ID: PM0079785
Poor rotamers = 44 (4.34%)
Ramachandran outliers = 66 (5.32%)
Ψ 0 Ramachandran favoured = 1036 (83.55%)
Ramachandran allowed = 94.7%
C𝛽 deviations > 0.25A = N.A
Residues with bad bonds = 0/4967 (0.00%)
Predicted model # 2 −180 Residues with bad angles = 66/6207 (1.06%)
−180 0 180
Φ
180
PMDB ID: PM0079786
Poor rotamers = 41 (4.04%)
Ramachandran outliers = 86 (6.94%)
Ψ 0 Ramachandran favoured = 999 (80.56%)
Ramachandran allowed = 93.1%
C𝛽 deviations > 0.25A = N.A
Residues with bad bonds = 1/4967 (0.02%)
Predicted model # 3 −180 Residues with bad angles = 53/6207 (0.85%)
−180 0 180
Φ
180
Predicted model # 4 PMDB ID: PM0079787
Poor rotamers = 35 (3.45%)
Ramachandran outliers = 148 (11.94%)
Ψ 0 Ramachandran favoured = 901 (72.6%)
Ramachandran allowed = 89.0%
C𝛽 deviations > 0.25A = N.A
Residues with bad bonds = 0/4967 (0.00%)
Residues with bad angles = 106/6207 (1.71%)
−180
−180 0 180
Φ
180
Predicted model # 5 PMDB ID: PM0079788
Poor rotamers = 39 (3.85%)
Ramachandran outliers = 137 (11.05%)
Ψ 0 Ramachandran favoured = 891 (71.85%)
Ramachandran allowed = 91.8%
C𝛽 deviations > 0.25A = N.A
Residues with bad bonds = 0/4967 (0.00%)
Residues with bad angles = 116/6207 (1.87%)
−180
−180 0 180
Φ

(a)
Human IRS-1 predicted Ramachandaran plots of Geometrical analysis of
structures by I-TASSER predicted structures predicted structures
Predicted model # 1 180
PMDB ID: PM0079789
Poor rotamers = 42 (4.08%)
Ramachandran outliers = 109 (8.16%)
Ψ 0 Ramachandran favoured = 1069 (80.01%)
Ramachandran allowed = 91.8%
C𝛽 deviations > 0.25 A = 84 (7.12%)
Residues with bad bonds = 0/5351 (0.00%)
Residues with bad angles = 80/6687 (1.20%)
−180
−180 0 180
Φ
180 PMDB ID: PM0079790
Predicted model # 2 Poor rotamers = 50 (4.85%)
Ramachandran outliers = 121 (9.06%)
Ψ Ramachandran favoured = 1029 (77.02%)
0 Ramachandran allowed = 90.9%
C𝛽 deviations > 0.25 A = 92 (7.80%)
Residues with bad bonds = 1/5351 (0.02%)
−180 Residues with bad angles = 102/6687 (0.81%)
−180 0 180
Φ
180
PMDB ID: PM0079791
Poor rotamers = 36 (3.50%)
Ramachandran outliers = 124 (9.28%)
Ψ 0 Ramachandran favoured = 1027 (76.8%)
Ramachandran allowed = 90.7%
C𝛽 deviations > 0.25A = 77 (6.53%)
Residues with bad bonds = 2/5351 (0.04%)
Predicted model # 3 Residues with bad angles = 116/6687 (1.73%)
−180
−180 0 180
Φ
180
Predicted model # 4 PMDB ID: PM0079792
Poor rotamers = 36 (3.50%)
Ramachandran outliers = 186 (13.92%)
Ψ 0 Ramachandran favoured = 924 (69.16%)
Ramachandran allowed = 86.1%
C𝛽 deviations > 0.25A = 65 (5.51%)
Residues with bad bonds = 0/5351 (0.00%)
Residues with bad angles = 149/6687 (2.2%)
−180
−180 0 180
Φ
180
Predicted model # 5 PMDB ID: PM0079793
Poor rotamers = 38 (3.69%)
Ramachandran outliers = 109 (8.16%)
Ψ 0 Ramachandran favoured = 1039 (77.7%)
Ramachandran allowed = 91.8%
C𝛽 deviations > 0.25A = 50 (4.24%)
Residues with bad bonds = 0/5351 (0.00%)
−180 Residues with bad angles = 78/6687 (1.17%)
−180 0 180
Φ

(b)

Figure 3: Homology models of human IRS-1 and IRS-2 retrieved through I-TASSER. Five models were predicted as possible 3D structures
for both IRS-1 and IRS-2 proteins. The best model for each protein was selected on the basis of Ramachandran plots and their geometrical
configuration. (a) Human IRS-1 full-length predicted 3D structures by I-TASSER with Ramachandran plots and geometry. (b) Human IRS-2
predicted 3D structures by I-TASSER with Ramachandran plots and geometry.
Advances in Bioinformatics
IRS-1
IRS-2
Identity = 4.02%
Similarity = 8.71%
Figure 4: Homology models of both human IRS-1 and IRS-1 were
retrieved utilizing automated protein modelling options through
I-TASSER. The best 3D representative models for both IRS-1 (in
red) and IRS-2 (in green) were aligned using FATCAT server and
viewed in Chimera software package. Sequence alignment revealed
that both proteins are significantly different in structure and only
have 4.02% identity and 8.71% similarity.
predicted kinases involved in phosphorylation of many serine
and threonine residues in IRS-2. INSR, syk, SRC, and insulin
receptor kinase are the most predicted kinases for tyrosine
phosphorylation as shown in Table 2S. Intrinsic tyrosine
kinase in the 𝛽-subunit of IR is known to phosphorylate
diverse substrates, including IRS-2 [51].
3.4.6. Prediction of O-𝛽-GlcNAc Modification and Potential
Yin Yang Sites for IRS-1. YinOYang prediction server showed
that the human IRS-1 has 57 potential sites for O-𝛽-GlcNAc
modifications (Figure 6(a)). It had been studied by various
researchers that phosphorylate Ser/Thr residues can also
undergo O-𝛽-GlcNAc modification when OGT and kinases
compete for same site modification [52–54], thus indicat-
ing a possibility for crosstalk between O-glycosylation and
phosphorylation on these residues. YinOYang 1.2 showed that
IRS-1 has high potential for O-glycosylation/phosphorylation
interplay (Table 3) and 37 possible yin yang sites were
predicted for the crosstalk of phosphorylation and O-𝛽-
GlcNAc modification (Figure 6(b)). These sites are Ser-7, 268,
270, 273, 303, 312, 341, 348, 388, 391, 393, 395, 413, 417,
543, 639, 680, 681, 683, 684, 741, 807, 808, 809, 985, 1000,
1005, 1011, 1035, 1036, 1101, 1105, 1213, and 1223 and Thr-305,
847, and 1004 (Table 3). Out of these 37 sites, 7 sites (Ser-
7, 680, 681, 683, 684, 1036, and 1213) were labeled as false
positive (FP) yin yang sites because they are nonconserved.
Meanwhile, some conserved Ser and Thr residues that were
not predicted to be O-𝛽-GlcNAc modified, but exhibited very
high phosphorylation potential, and also close to threshold
value of O-glycosylation, are named as false negative (FN) yin
11
yang sites. These residues include Ser-383, 412, 1037 and Thr-
387.
3.4.7. Prediction of O-𝛽-GlcNAc Modification and Potential
Yin Yang Sites for IRS-2. According to YinOYang 1.2 results,
IRS-2 had high potential for O-𝛽-GlcNAc modification and
64 Ser/Thr residues were predicted to be O-glycosylated
(Figure 7(a)). As described earlier, these sites can also
undergo phosphorylation modification that may lead to
interplay between O-glycosylation and phosphorylation on
these sites. YinOYang 1.2 predicted 40 possible yin yang sites
(Figure 7(b)). These sites were Ser-304, 308, 311, 384, 400, 428,
438, 444, 449, 482, 483, 485, 489, 523, 560, 606, 620, 665, 731,
736, 953, 956, 967, 984, 988, 1012, 1022, 1024, 1026, 1027, 1048,
1149, 1162, and 1203 and Thr-443, 777, 844, 965, 1157, and 1159
(Table 4). Ser-162, 342, 357, 447, 714, 848, 946, 966, and 1115,
and Thr-713 and 1202 were predicted as FN-yin yang sites.
4. Discussion
Insulin is a primary hormone that regulates glucose transport
and homeostasis in cell systems through its interaction with
its receptors (IR) via tyrosine autophosphorylation of IR.
Activation of IR subsequently phosphorylates its substrates
(IRSs). Insulin receptor substrates 1 and 2 are specific sub-
strates for IR/IGFR and widely expressed in mammalian
tissues. Both IRS 1 and 2 contain phosphotyrosine-binding
(PTB) domains. Tyrosine phosphorylation of IRS proteins is
well studied and more than 20 potential tyrosine phospho-
rylation sites are mapped on IRSs. During insulin signaling,
IRS proteins phosphorylate and activate AKT/PKB/MAPK
pathways that regulate insulin mediated metabolic actions
and control cell growth and differentiation [10, 20].
Despite Tyr phosphorylation, IRSs also phosphorylate
on Ser/Thr residues and phosphorylation on these residues
may effect downstream insulin signaling. More than 30
Ser/Thr residues on IRS-1 and 50 on IRS-2 have been mapped
using different analytical techniques. However, function of all
mapped Ser/Thr residues on IRSs is still undetermined. Our
results showed that both IRS-1 and IRS-2 have high potential
to be phosphorylated on Ser and Thr residues and more
than 100 Ser/Thr residues have been predicted including
previously mapped residues on IRS 1 and 2 on both N- and
C-terminals including PTB domain (Figures 5(a) and 5(b)).
These predicted residues are conserved as shown in Figures
1 and 2 (multiple alignment of IRS-1 and IRS-2, resp.) and
may have potential to modify IRSs functioning. Meanwhile
various kinases such as GSK3, PKC, PKA, ERK, mTOR, S6K,
PKG, CDK5, CKII, and JNK are involved in Ser/Thr phos-
phorylation of IRSs. Both ERK and mTOR are experimentally
verified kinases that can cause phosphorylation at Ser-616
[55]. Phosphorylation of Ser-312 is mediated by increased
activation of kinases downstream of PI3-kinase, comprising
S6K1, IKK𝛽, mTOR, PKC, and JNK [22, 25, 56]. Akt-mTOR
activity is highly activated within neurons in many AD cases
[57]. S6K activation was also found to be induced in AD that
induces phosphorylation of Ser-636 and 639 of IRS-1 [58].
Ser-312, 616, 636, 639, and 1101 in IRS-1 are experimentally
12 Advances in Bioinformatics

NetPhos 2.0 predicted phosphorylation sites in sp P35568-I NetPhos 2.0 predicted phosphorylation sites in sp Q9Y4H2-I
Phosphorylation

Phosphorylation
1 1
potential

potential
0 0
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
Sequence position Sequence position

Serine Tyrosine Serine Tyrosine

Threonine Threshold Threonine Threshold
(a) (b)

Figure 5: Graphical representation of the potential Ser, Thr, and Tyr residues for phosphorylation sites in human IRS-1 and IRS-2. (a)
Predicted potential sites for phosphorylation on Ser, Thr, and Tyr residues. The light gray horizontal line shows the threshold for modification
potential. The blue, green, and red vertical lines indicate the potential phosphorylated Ser, Thr, and Tyr residues, respectively. This graph was
generated by NetPhos 2.0 web based program. (b) Predicted potential sites for phosphorylation on Ser, Thr, and Tyr residues. The light gray
horizontal line shows the threshold for modification potential. The blue, green, and red vertical lines indicate the potential phosphorylated
Ser, Thr, and Tyr residues, respectively. This graph was generated by NetPhos 2.0 web based program.

YinOYang predicted O-𝛽-GlcNAc sites in sp P35568-I YinOYang predicted O-𝛽-GlcNAc sites in sequence
O-Glycosylation
O-Glycosylation

1
potential

potential 1

0 0
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
Sequence position Sequence position

O-GlcNAc potential O-GlcNAc potential Yin-Yang

Threshold Threshold
(a) (b)

Figure 6: Graphical representation of the potential Ser and Thr residues for O-𝛽-GlcNAc as well as yin yang sites in human IRS-1. (a) Predicted
potential sites for O-𝛽-GlcNAc modification of Ser and Thr. Green vertical line shows O-𝛽-GlcNAc modification potential of Ser/Thr residues,
while the light blue wavy line indicates the threshold for modification potential. YinOYang 1.2 web based program generated this graph. A
total number of 57 potential Ser/Thr residues showed O-𝛽-GlcNAc modification potential including Ser-7, 268, 270, 273, 295, 303, 312, 341,
348, 388, 391, 393, 394, 395, 412, 413, 417, 440, 541, 543, 624, 639, 641, 680, 681, 682, 683, 684, 741, 807, 808, 809, 917, 985, 1000, 1005, 1011,
1025, 1035, 1036, 1037, 1101, 1105, 1213, and 1223 and Thr-305, 311, 351, 387, 539, 743, 803, 847, 851, 1004, 1030, and 1045. (b) Predicted yin yang
residues for human IRS-1. A total of 37 Ser/Thr residues showed potential for possible yin yang sites for phosphorylation and O-glycosylation
interplay.

YinOYang predicted O-𝛽-GlcNAc sites in sp Q9Y4H2-I YinOYang predicted O-𝛽-GlcNAc sites in sequence
O-Glycosylation
O-Glycosylation

1
potential

potential

0 0
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
Sequence position Sequence position

O-GlcNAc potential O-GlcNAc potential Yin-Yang

Threshold Threshold
(a) (b)

Figure 7: Graphical representation of the potential Ser and Thr residues for O-𝛽-GlcNAc as well as yin yang sites in human IRS-2. (a) Predicted
potential sites for O-𝛽-GlcNAc modification of Ser and Thr. Green vertical line shows O-𝛽-GlcNAc modification potential of Ser/Thr residues,
while the light blue wavy line indicates the threshold for modification potential. YinOYang 1.2 web based program generated this graph. A
total number of 64 potential Ser/Thr residues showed O-𝛽-GlcNAc modification potential including Ser-3, 162, 304, 308, 311, 357, 384, 400,
428, 438, 444, 449, 482, 483, 485, 489, 523, 560, 591, 592, 606, 615, 619, 620, 665, 679, 684, 704, 714, 731, 736, 848, 941, 944, 945, 946, 952,
953, 956, 966, 967, 984, 988, 1012, 1022, 1024, 1026, 1027, 1048, 1103, 1149, 1162, and 1203 and Thr-344, 443, 713, 719, 777, 844, 965, 1157, 1159,
1202, and 1319. (b) Predicted yin yang residues for human IRS-1. A total of 41 Ser/Thr residues showed potential for possible yin yang sites for
phosphorylation and O-glycosylation interplay.
Advances in Bioinformatics 13

Table 3: Experimental and predicted O-𝛽-GlcNAc and yin yang sites in human IRS-1.

Substrate O-𝛽-GlcNAc status

Yin yang residues
Residues PS Conservation SA YinOYang 1.2
Ser 7 NC E ++ Y(FP)
Ser 268 CS B ++ Y
Ser 270 C E ++ Y
Ser 273 C B + Y
Ser 295 C E + −
Ser 303 CS E + Y
Thr 305 C E + Y
Thr 311 C E + −
Ser 312 C B + Y
Ser 341 SC E + Y
Ser 348 C E + Y
Thr 351 CS E + −
Ser 383 C E − Y(FN)
Thr 387 C E +++ Y(FN)
Ser 388 C E ++ Y
Ser 391 C E ++++ Y
Ser 393 C B + Y
Ser 394 C B + −
Ser 395 C E + Y
Ser 412 C E +++ Y(FN)
Ser 413 C E ++ Y
Ser 417 C E ++ Y
Ser 440 C B ++ −
Thr 539 C E ++ −
Ser 541 C E + −
Ser 543 C E + Y
Ser 624 NC E + −
Ser 639 C E + Y
Ser 641 C E + −
Ser 680 NC E ++ Y(FP)
Ser 681 NC E +++ Y(FP)
Ser 682 NC E ++ −
Ser 683 NC E + Y(FP)
Ser 684 NC E + Y(FP)
Ser 741 C E + Y
Thr 743 C E ++ −
Thr 803 NC E ++ −
Ser 807 C E + Y
Ser 808 C E +++ Y
Ser 809 C E ++ Y
Thr 847 C E + Y
Thr 851 NC E + −
Ser 917 NC E + −
Ser 985 SC E + Y
Ser 1000 SC E + Y
Thr 1004 SC E + Y
14 Advances in Bioinformatics

Table 3: Continued.
Substrate O-𝛽-GlcNAc status
Yin yang residues
Residues PS Conservation SA YinOYang 1.2
Ser 1005 SC E + Y
Ser 1011 CS E ++ Y
Ser 1025 SC E + −
Thr 1030 SC E + −
Ser 1035 SC E + Y
Ser 1036 NC E +++ Y(FP)
Ser 1037 CS E ++++ Y(FN)
Thr 1045 NC E + −
Ser 1101 C E + Y
Ser 1105 C E ++ Y
Ser 1213 NC E + Y(FP)
Ser 1223 C B ++ Y
PS: position; SA: surface accessibility; E: exposed; B, buried; C: conserved; NC: nonconserved; CS: conserved substitutions; SC: semiconserved; Y: predicted as
yin yang sites by YinOYang 1.2; FP: false positive; FN: false negative; −: negative/not determined yet; +: positive; ++: highly positive; +++: very highly positive;
++++: very very highly positive.

verified phosphorylation sites. Phosphorylation of Ser-312
causes IRS-1 proteasomal degradation that leads to insulin
resistance under both pathological and physiological condi-
tions, while phosphorylation of Ser-616 is supposed to inhibit
insulin activation of PI3-kinase. Furthermore, phosphoryla-
tion on Ser-1101 by S6K1 on C-terminal of IRS-1 is thought
to be involved in blocking tyrosine as well as AKT phospho-
rylation that leads to insulin resistance in mice as well as in
human [21, 23, 25]. GSK-3 and JNK sequentially phospho-
rylate IRS-2 at serine residues and its phosphorylation plays
inhibitory role in hepatic insulin signaling [51, 58, 59].
The O-𝛽-GlcNAc modification is known to be influ-
ential and analogous to phosphorylation. Some researchers
indicated increased phosphorylation and reduced O-GlcNAc
levels in the AD and diabetes patients [60–62]. These findings
made O-glycosylation as important PTM in elucidating the
mechanism of such metabolic disorders. In this study we used
YinOYang 1.2 server to predict O-glycosylation on Ser/Thr
residues of both IRS-1 and 2, and more than 50 Ser/Thr
residues were shown to have high potential for O-𝛽-GlcNAc
modification on IRS-1 (Figures 6(a) and 7(a) and Tables 3
and 4). O-𝛽-GlcNAc modification in human IRS-1 has been
reported at Ser-984 or 985, 1011, and possibly at multiple
residues within 1025–1045 [33]. Based on our results Ser-
984 is more prone to be O-glycosylated than Ser-985 while
Ser-1030, 1035, 1037 and Thr-1045 have potential to be O-
glycosylated in 1025–1045 sequence on the basis of conserva-
tion. Meanwhile western blotting and site-directed mutagen-
esis studies in rat suggested Ser-1036 (human numbering Ser-
1037) on IRS-1 as possible site of O-𝛽-GlcNAc modification.
Ser-1037 is conserved in human IRS-1 and identification of
this site will be helpful in exploring the biological implication
of the O-𝛽-GlcNAc modification [33]. Although to date no
O-glycosylated residue is mapped on IRS-2, experimental
data showed that IRS-2 also have potential to be glycosylated
as IRS-1 and this glycosylation was shown to be reduced in
diabetes and AD [32]. In this study we have predicted more
than 65 Ser/Thr residues on IRS-2 that can act as potential
O-glycosylated sites and may ease researchers to map correct
O-glycosylated residues on IRS-2.
It is established fact that O-𝛽-GlcNAc can mimic or
inhibit phosphorylation on the neighboring or same residues
and if this interplay happened on same Ser/Thr residues these
sites are called yin yang residues. These yin yang residues
can be used as therapeutic targets to overcome different
diseases [42, 52]. Protein 3D structures are best models to
tell whether the predicted residues are accessible or not for
these types of interplays [27, 41]. However, for both IRS-1
and IRS-2, complete 3D have not been determined yet, so we
used ab initio models to design the complete 3D structures
of both proteins. As both IRS-1 and IRS-2 are involved in
insulin signaling mechanism, best possible 3D structures
for both proteins were selected on basis of Ramachandran
plots (Figures 3(a) and 3(b)) and were aligned together to
be compared with each other (Figure 4). We found that
both proteins have significantly nonidentical structures as
their secondary structure previously described. To determine
yin yang sites and surface accessibility, we predicted surface
accessible residues on the basis of their secondary structure
by using NetPhos P server (as given in Table S1 and S2
for both IRS-1 and IRS-2, resp.). Secondary structure based
prediction method showed that both proteins have high
accessibility for PTMs on Ser/Thr residues except Ser-268,
273, 312, 393, 394, 440, 1223 on IRS-1, Ser-428, and Thr-
344. Although secondary structure based prediction method
showed no/reduced surface accessibility for Ser-312 of IRS-1,
predicted 3D structure revealed that Ser-312 is accessible for
PTMs (Figure 8). Moreover, Ser-312 is experimentally verified
and known phosphorylation residue, which confirms the
exposed accessibility of this residue. Ser-312 phosphorylation
Advances in Bioinformatics 15

Table 4: Experimental and predicted O-𝛽-GlcNAc and yin yang sites in human IRS-2.

Substrate O-𝛽-GlcNAc status

Yin yang residues
Residues PS Conservation SA YinOYang 1.2
Ser 3 C E + −
Ser 162 C E ++ Y(FN)
Ser 304 C E ++ Y
Ser 308 C E + Y
Ser 311 C E + Y
Ser 342 C E − Y(FN)
Thr 344 C B + −
Ser 357 CS E ++ Y(FN)
Ser 384 C E ++++ Y
Ser 400 C E + Y
Ser 428 C B + Y
Ser 438 C E + Y
Thr 443 C E ++++ Y
Ser 444 C E ++ Y
Ser 447 C E − Y(FN)
Ser 449 C E + Y
Ser 482 C E + Y
Ser 483 C E ++++ Y
Ser 485 C E + Y
Ser 489 C E + Y
Ser 523 C E + Y
Ser 560 C E + Y
Ser 591 C E + −
Ser 592 C E + −
Ser 606 C E + Y
Ser 615 C E + −
Ser 619 C E + −
Ser 620 C E + Y
Ser 665 SC E ++ Y
Ser 679 C E + −
Ser 684 C E + −
Ser 704 SC E + −
Thr 713 SC E ++ Y(FN)
Ser 714 C E ++ Y(FN)
Thr 719 C E + −
Ser 731 C E + Y
Ser 736 C E + Y
Thr 777 CS E + Y
Thr 844 C E ++ Y
Ser 848 CS E ++ Y(FN)
Ser 941 C E + −
Ser 944 C E + −
Ser 945 C E + −
Ser 946 C E ++++ Y(FN)
Ser 952 C E + −
Ser 953 C E ++ Y
16 Advances in Bioinformatics

Table 4: Continued.
Substrate O-𝛽-GlcNAc status
Yin yang residues
Residues PS Conservation SA YinOYang 1.2
Ser 956 C E + Y
Thr 965 C E ++ Y
Ser 966 C E +++ Y(FN)
Ser 967 C E + Y
Ser 984 C E + Y
Ser 988 C E + Y
Ser 1012 C E + Y
Ser 1022 C E +++ Y
Ser 1024 C E ++ Y
Ser 1026 C E + Y
Ser 1027 C E ++ Y
Ser 1048 CS E + Y
Ser 1103 C E + −
Ser 1115 C E − Y(FN)
Ser 1149 C E + Y
Thr 1157 C E + Y
Thr 1159 C E + Y
Ser 1162 C E ++ Y
Thr 1202 C E ++ Y(FN)
Ser 1203 C E ++ Y
Thr 1319 C B + −
PS: position; SA: surface accessibility; E: exposed; B: buried; C: conserved; CS: conserved substitutions; SC: semiconserved; Y: predicted as yin yang sites by
YinOYang 1.2; FN: false negative; −: negative/not determined yet; +: positive; ++: highly positive; +++: very highly positive; ++++: very very highly positive.

is important in insulin resistance and has negative regulation

of insulin signaling. Interestingly, Ser-312 is situated very near
to PTB domain (Figure 8) and any change in its vicinity
via PTMs should alter surrounding areas [21]. Furthermore
phosphorylation on three important Ser residues 984, 1037, Ser-1037
and 1101 in C-terminal of IRS-1 also has been shown to induce Ser-312 Ser-984
insulin resistance [33]. All these residues were conserved, Ser-1101
accessible for such modifications based on 3D structure, and
predicted as yin yang residues in our study. This finding
showed that alternative O-glycosylation on these residues
Figure 8: Space filled model of human IRS-1 protein showing higher
can inhibit the protein functioning rise by phosphorylation.
surface accessibility of possible therapeutic yin yang residues for
Although a lot of phosphorylation sites mapped on IRS-2 interplay between phosphorylation and O-glycosylation. Blue and
like Ser-303, 343, 675, and 907 are thought to be involved green colours denote the Ser and Thr residues, respectively.
in inducing insulin resistance, we were unable to predict O-
glycosylation on these residues due to limited experimental
data availability. On the other hand, we found more than 40
Ser/Thr residues on IRS-2 that has high potential to act as
possible yin yang sites (Table 4), and many of them may act Conflict of Interests
as therapeutic targets in future. The authors have no conflict of interests regarding the
Here we propose that alternative O-glycosylation on publication of this paper.
Ser-312 at N-terminal and Ser-984, Ser-1037, and Ser-1101
at C-terminal of IRS-1 is an important modification that
becomes reduced in diabetes and AD due to impaired glucose Authors’ Contribution
metabolism. During normal circumstances, O-glycosylation
can block phosphorylation on these residues and can act as Zainab Jahangir and Waqar Ahmad contributed equally to
possible therapeutic sites to reduce risk of diabetes and AD. this work.
Advances in Bioinformatics
References
[1] D. J. Selkoe, “Alzheimer’s disease: genotypes, phenotype, and
treatments,” Science, vol. 275, no. 5300, pp. 630–631, 1997.
[2] Alzheimer’s Association, “2012 Alzheimer’s disease facts and
figures,” Alzheimer’s & Dementia, vol. 8, no. 2, pp. 131–168, 2012.
[3] W. H. Gispen and G.-J. Biessels, “Cognition and synaptic
plasticity in diabetes mellitus,” Trends in Neurosciences, vol. 23,
no. 11, pp. 542–549, 2000.
[4] S. Hoyer, “Is sporadic Alzheimer disease the brain type of non-
insulin dependent diabetes mellitus? A challenging hypothesis,”
Journal of Neural Transmission, vol. 105, no. 4-5, pp. 415–422,
1998.
[5] S. M. de la Monte and J. R. Wands, “Alzheimer’s disease is type
3 diabetes-evidence reviewed,” Journal of Diabetes Science and
Technology, vol. 2, no. 6, pp. 1101–1113, 2008.
[6] W. Ahmad, “Overlapped metabolic and therapeutic links
between Alzheimer and diabetes,” Molecular Neurobiology, vol.
47, no. 1, pp. 399–424, 2013.
[7] M.-K. Sun and D. L. Alkon, “Links between Alzheimer’s disease
and diabetes,” Drugs of Today, vol. 42, no. 7, pp. 481–489, 2006.
[8] Y. Deng, B. Li, Y. Liu, K. Iqbal, I. Grundke-Iqbal, and C.-X.
Gong, “Dysregulation of insulin signaling, glucose transporters,
𝑂-GlcNAcylation, and phosphorylation of tau and neurofila-
ments in the brain: implication for Alzheimer’s disease,” The
American Journal of Pathology, vol. 175, no. 5, pp. 2089–2098,
2009.
[9] Y. D. Ke, F. Delerue, A. Gladbach, J. Götz, and L. M. Ittner,
“Experimental diabetes mellitus exacerbates Tau pathology in
a transgenic mouse model of Alzheimer’s disease,” PLoS ONE,
vol. 4, no. 11, Article ID e7917, 2009.
[10] G. Sesti, M. Federici, M. L. Hribal, D. Lauro, P. Sbraccia, and R.
Lauro, “Defects of the insulin receptor substrate (IRS) system in
human metabolic disorders,” FASEB Journal, vol. 15, no. 12, pp.
2099–2111, 2001.
[11] D. J. Withers, D. J. Burks, H. H. Towery, S. L. Altamuro, C.
L. Flint, and M. F. White, “Irs-2 coordinates Igf-1 receptor-
mediated 𝛽-cell development and peripheral insulin signalling,”
Nature Genetics, vol. 23, no. 1, pp. 32–40, 1999.
[12] D. J. Withers, J. S. Gutierrez, H. Towery et al., “Disruption of
IRS-2 causes type 2 diabetes in mice,” Nature, vol. 391, no. 6670,
pp. 900–904, 1998.
[13] C. Selman, S. Lingard, A. I. Choudhury et al., “Evidence
for lifespan extension and delayed age-related biomarkers in
insulin receptor substrate 1 null mice,” FASEB Journal, vol. 22,
no. 3, pp. 807–818, 2008.
[14] A. Taguchi, L. M. Wartschow, and M. F. White, “Brain IRS2 sig-
naling coordinates life span and nutrient homeostasis,” Science,
vol. 317, no. 5836, pp. 369–372, 2007.
[15] M. Schubert, D. P. Brazil, D. J. Burks et al., “Insulin receptor
substrate-2 deficiency impairs brain growth and promotes tau
phosphorylation,” The Journal of Neuroscience, vol. 23, no. 18,
pp. 7084–7092, 2003.
[16] A. M. Moloney, R. J. Griffin, S. Timmons, R. O’Connor, R. Ravid,
and C. O’Neill, “Defects in IGF-1 receptor, insulin receptor and
IRS-1/2 in Alzheimer’s disease indicate possible resistance to
IGF-1 and insulin signalling,” Neurobiology of Aging, vol. 31, no.
2, pp. 224–243, 2010.
[17] R. K. Dearth, X. Cui, H.-J. Kim, D. L. Hadsell, and A. V. Lee,
“Oncogenic transformation by the signaling adaptor proteins
insulin receptor substrate (IRS)-1 and IRS-2,” Cell Cycle, vol. 6,
no. 6, pp. 705–713, 2007.
17
[18] M. Mann and O. N. Jensen, “Proteomic analysis of post-
translational modifications,” Nature Biotechnology, vol. 21, no.
3, pp. 255–261, 2003.
[19] S. A. Summers, “Ceramides in insulin resistance and lipotoxic-
ity,” Progress in Lipid Research, vol. 45, no. 1, pp. 42–72, 2006.
[20] G. Sesti, “Pathophysiology of insulin resistance,” Best Practice
and Research: Clinical Endocrinology and Metabolism, vol. 20,
no. 4, pp. 665–679, 2006.
[21] M. W. Greene, H. Sakaue, L. Wang, D. R. Alessi, and R. A.
Roth, “Modulation of insulin-stimulated degradation of human
insulin receptor substrate-1 by serine 312 phosphorylation,” The
Journal of Biological Chemistry, vol. 278, no. 10, pp. 8199–8211,
2003.
[22] R. J. Griffin, A. Moloney, M. Kelliher et al., “Activation of
Akt/PKB, increased phosphorylation of Akt substrates and
loss and altered distribution of Akt and PTEN are features of
Alzheimer’s disease pathology,” Journal of Neurochemistry, vol.
93, no. 1, pp. 105–117, 2005.
[23] L. Rui, V. Aguirre, J. K. Kim et al., “Insulin/IGF-1 and TNF-
𝛼 stimulate phosphorylation of IRS-1 at inhibitory Ser307 via
distinct pathways,” The Journal of Clinical Investigation, vol. 107,
no. 2, pp. 181–189, 2001.
[24] Y. Zick, “Ser/Thr phosphorylation of IRS proteins: a molecular
basis for insulin resistance,” Science’s STKE, vol. 2005, no. 268,
p. pe4, 2005.
[25] H. Yu and T. Rohan, “Role of the insulin-like growth factor
family in cancer development and progression,” Journal of the
National Cancer Institute, vol. 92, no. 18, pp. 1472–1489, 2000.
[26] S. M. Firth and R. C. Baxter, “Cellular actions of the insulin-like
growth factor binding proteins,” Endocrine Reviews, vol. 23, no.
6, pp. 824–854, 2002.
[27] W. Ahmad, K. Shabbiri, B. Ijaz et al., “Serine 204 phosphory-
lation and O - GlcNAC interplay of IGFBP-6 as therapeutic
indicator to regulate IGF-II functions in viral mediated hepa-
tocellular carcinoma,” Virology Journal, vol. 8, article 208, 2011.
[28] C. H. Wu, R. Apweiler, A. Bairoch et al., “The Universal
Protein Resource (UniProt): an expanding universe of protein
information,” Nucleic Acids Research, vol. 34, pp. D187–D191,
2006.
[29] N. E. Zachara and G. W. Hart, “The emerging significance of O-
GlcNAc in cellular regulation,” Chemical Reviews, vol. 102, no.
2, pp. 431–438, 2002.
[30] M. F. White, “Regulating insulin signaling and 𝛽-cell function
through IRS proteins,” Canadian Journal of Physiology and
Pharmacology, vol. 84, no. 7, pp. 725–737, 2006.
[31] K. F. Petersen and G. I. Shulman, “Etiology of insulin resistance,”
American Journal of Medicine, vol. 119, no. 5, 2006.
[32] C. D’Alessandris, F. Andreozzi, M. Federici et al., “Increased
O-glycosylation of insulin signaling proteins results in their
impaired activation and enhanced susceptibility to apoptosis in
pancreatic 𝛽-cells,” The FASEB Journal, vol. 18, no. 9, pp. 959–
961, 2004.
[33] A. L. Kleini, M. N. Berkaw, M. G. Buse, and L. E. Ball, “𝑂-
linked 𝑁-acetylglucosamine modification of insulin receptor
substrate-1 occurs in close proximity to multiple SH2 domain
binding motifs,” Molecular and Cellular Proteomics, vol. 8, no.
12, pp. 2733–2745, 2009.
[34] L. E. Ball, M. N. Berkaw, and M. G. Buse, “Identification of the
major site of O-linked 𝛽-N-acetylglucosamine modification in
the C terminus of insulin receptor substrate-1,” Molecular and
Cellular Proteomics, vol. 5, no. 2, pp. 313–323, 2006.
 18
[35] N. Eswar, B. Webb, M. A. Marti-Renom et al., “Unit 5.6. Chapter
5. Comparative protein structure modeling using modeller,” in
Current Protocols in Bioinformatics, 2006.
[36] Y. Zhang, “I-TASSER server for protein 3D structure predic-
tion,” BMC Bioinformatics, vol. 9, article 40, 2008.
[37] K. Arnold, L. Bordoli, J. Kopp, and T. Schwede, “The SWISS-
MODEL workspace: a web-based environment for protein
structure homology modelling,” Bioinformatics, vol. 22, no. 2,
pp. 195–201, 2006.
[38] I. W. Davis, L. W. Murray, J. S. Richardson, and D. C. Richard-
son, “MolProbity: structure validation and all-atom contact
analysis for nucleic acids and their complexes,” Nucleic Acids
Research, vol. 32, pp. W615–W619, 2004.
[39] E. F. Pettersen, T. D. Goddard, C. C. Huang et al., “UCSF
Chimera—a visualization system for exploratory research and
analysis,” Journal of Computational Chemistry, vol. 25, no. 13,
pp. 1605–1612, 2004.
[40] P. Baldi and S. Brunak, Bioinformatics: The Machine Learning
Approach, MIT Press, Cambridge, Mass, USA, 2nd edition,
2001.
[41] W. Ahmad, K. Shabbiri, B. Ijaz et al., “Claudin-1 required for
HCV virus entry has high potential for phosphorylation and O-
glycosylation,” Virology Journal, vol. 8, article 229, 2011.
[42] W. Ahmad, K. Shabbiri, N. Nazar, S. Nazar, S. Qaiser, and M.
A. S. Mughal, “Human linker histones: interplay between phos-
phorylation and O-𝛽-GlcNAc to mediate chromatin structural
modifications,” Cell Division, vol. 6, article 15, 2011.
[43] N. Blom, S. Gammeltoft, and S. Brunak, “Sequence and
structure-based prediction of eukaryotic protein phosphoryla-
tion sites,” Journal of Molecular Biology, vol. 294, no. 5, pp. 1351–
1362, 1999.
[44] Z. Songyang, S. Blechner, N. Hoagland, M. F. Hoekstra, H.
Piwnica-Worms, and L. C. Cantley, “Use of an oriented peptide
library to determine the optimal substrates of protein kinases,”
Current Biology, vol. 4, no. 11, pp. 973–982, 1994.
[45] N. Blom, T. Sicheritz-Pontén, R. Gupta, S. Gammeltoft, and
S. Brunak, “Prediction of post-translational glycosylation and
phosphorylation of proteins from the amino acid sequence,”
Proteomics, vol. 4, no. 6, pp. 1633–1649, 2004.
[46] H.-D. Huang, T.-Y. Lee, S.-W. Tzeng, and J.-T. Horng,
“KinasePhos: a web tool for identifying protein kinase-specific
phosphorylation sites,” Nucleic Acids Research, vol. 33, no. 2, pp.
W226–W229, 2005.
[47] F. Diella, S. Cameron, C. Gemünd et al., “Phospho.ELM: a
database of experimentally verified phosphorylation sites in
eukaryotic proteins,” BMC Bioinformatics, vol. 5, article 79,
2004.
[48] R. Gupta and S. Brunak, “Prediction of glycosylation across
the human proteome and the correlation to protein function,”
Pacific Symposium on Biocomputing, pp. 310–322, 2002.
[49] B. Petersen, T. N. Petersen, P. Andersen, M. Nielsen, and C.
Lundegaard, “A generic method for assignment of reliability
scores applied to solvent accessibility predictions,” BMC Struc-
tural Biology, vol. 9, article 51, 2009.
[50] J. C. Obenauer, L. C. Cantley, and M. B. Yaffe, “Scansite 2.0:
proteome-wide prediction of cell signalling interactions using
short sequence motifs,” Nucleic Acids Research, vol. 31, no. 13,
pp. 3635–3641, 2003.
[51] H. Sharfi and H. Eldar-Finkelman, “Sequential phosphorylation
of insulin receptor substrate-2 by glycogen synthase kinase-3
and c-Jun NH2-terminal kinase plays a role in hepatic insulin
Advances in Bioinformatics
signaling,” American Journal of Physiology: Endocrinology and
Metabolism, vol. 294, no. 2, pp. E307–E315, 2008.
[52] I. Ahmad, D. C. Hoessli, E. Walker-Nasir, S. M. Rafik, and A. R.
Shakoori, “Oct-2 DNA binding transcription factor: functional
consequences of phosphorylation and glycosylation,” Nucleic
Acids Research, vol. 34, no. 1, pp. 175–184, 2006.
[53] A. Kaleem, D. C. Hoessli, I. Ahmad et al., “Immediate-early gene
regulation by interplay between different post-translational
modifications on human histone H3,” Journal of Cellular Bio-
chemistry, vol. 103, no. 3, pp. 835–851, 2008.
[54] A. Kaleem, D. C. Hoessli, I.-U. Haq et al., “CREB in long-
term potentiation in hippocampus: role of post-translational
modifications-studies in silico,” Journal of Cellular Biochemistry,
vol. 112, no. 1, pp. 138–146, 2011.
[55] Z. Gao, D. Hwang, F. Bataille et al., “Serine phosphorylation
of insulin receptor substrate 1 by inhibitor 𝜅B kinase complex,”
Journal of Biological Chemistry, vol. 277, no. 50, pp. 48115–48121,
2002.
[56] T. Haruta, T. Uno, J. Kawahara et al., “A rapamycin-sensitive
pathway down-regulates insulin signaling via phosphorylation
and proteasomal degradation of insulin receptor substrate-1,”
Molecular Endocrinology, vol. 14, no. 6, pp. 783–794, 2000.
[57] W.-L. An, R. F. Cowburn, L. Li et al., “Up-regulation of
phosphorylated/activated p70 S6 kinase and its relationship to
neurofibrillary pathology in Alzheimer’s disease,” The American
Journal of Pathology, vol. 163, no. 2, pp. 591–607, 2003.
[58] S. S. Neukamm, R. Toth, N. Morrice et al., “Identification of the
Amino acids 300-600 of IRS-2 as 14-3-3 binding region with the
importance of IGF-1/insulin-regulated phosphorylation of Ser-
573,” PLoS ONE, vol. 7, no. 8, Article ID e43296, 2012.
[59] R. Zhande, W. Zhang, Y. Zheng et al., “Dephosphorylation by
default, a potential mechanism for regulation of insulin receptor
substrate-1/2, Akt, and ERK1/2,” The Journal of Biological Chem-
istry, vol. 281, no. 51, pp. 39071–39080, 2006.
[60] F. Liu, J. Shi, H. Tanimukai et al., “Reduced O-GlcNAcylation
links lower brain glucose metabolism and tau pathology in
Alzheimer’s disease,” Brain, vol. 132, no. 7, pp. 1820–1832, 2009.
[61] Y. Yu, L. Zhang, X. Li et al., “Differential effects of an O-
GlcNAcase inhibitor on tau phosphorylation,” PLoS ONE, vol.
7, no. 4, Article ID e35277, 2012.
[62] S. A. Yuzwa, X. Shan, M. S. MacAuley et al., “Increasing O-
GlcNAc slows neurodegeneration and stabilizes tau against
aggregation,” Nature Chemical Biology, vol. 8, no. 4, pp. 393–399,
2012.
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